Revision as of 15:38, 17 October 2016

Notebook

Story Began
Molecular Cloning
Cell Experiment
In Vitro Confirmation
Animal Experiment
Submission

Story Began
2015.11
2015.12
2016.01
2016.01~02
2016.02.22
2016.02~03
2016.03.04
2016.03.11
2016.03.15
2016.04~05
2016.06.05
Molecular Cloning
2016.06.05-12
2016.06.13-19
2016.06.20-26
2016.06.27-07.03
2016.07.04-10
2016.07.11-17
2016.07.18-24
2016.07.25-31
2016.08.01-07
2016.08.08-14
Cell Experiment
2016.08.15-21
2016.08.22-28
2016.08.29-09.04
In Vitro Confirmation
2016.09.05-11
2016.09.12-18
2016.09.19-25
Animal Experiment
2016.09.05
2016.09.07
2016.09.11
2016.09.12
2016.09.13
2016.09.14
2016.09.15-22
2016.09.16-21
2016.09.17
2016.09.17-24
2016.09.25
2016.09.28-10.05
Submission
2016.09.26-29
2016.10.04-09
2016.10.09-15

Since it is the first time for our school to participate in the iGEM competition ……
Having basic knowledge of iGEM, six kindred spirits gathered together and decided to participate in this competition. After deliberation, we started to prepare our team.
Our team, named as SYSU-MEDICINE, was successfully built, comprising four groups for lab work, modeling, art design and human practice respectively.
Assigned the tasks for winter vacation, including exploring feasible project theme, learned basic methods of literature search and summarized previous iGEM projects of the latest three years.
Six internal meetings were held and had a meet up with SYSU-China, from which we learned the importance of having a round knowledge of the rules of iGEM competition.
Started raising fund and lab resources.
Got in touch with leadership of Zhongshan School of Medicine and several professors and finally gained official support of Zhongshan School of Medicine. Refined our project at the suggestion of professors.
Final determination of supporting lab and fund resource.
Determined our first PI, second PI and instructor, to whom we presented our primary project and had a further discussion.
Signed up for iGEM.
Internal meetings for routine progress report were held, in which we decided the main topic of our project: mesenchymal stem cells (MSCs). After related article readings and with the guidance of our instructor, we further enriched our project and detailed our experiment plans. On May 20th, our final design was born.
Experiments begin.
Molecular Cloning
1. Extraction, Purification, and Analysis of total RNA from PBMCs (peripheral blood mononuclear cells)
2. RT-PCR——DNA : RNA hybridization
3. PCR, agarose gel electrophoresis, (primers without attB * cDNA; new primers with attB * cDNA) 10μl

100bp marker positive control 100bp marker only
4. Extraction, Purification, and Analysis of total RNA from new PBMCs (peripheral blood mononuclear cells) and RT-PCR——DNA : RNA hybridization
1. TIANprep Mini Plasmid Kit (TIANGEN)
2. Continue experiment yesterday.
PCR (new primers without attB * template cDNA; new primers without attB * template cDNA; CXCR5 primer * CXCR5 plasmid DNA), agarose gel electrophoresis 10μl

3. PCR
(new primers without attB * new template cDNA→ CXCR5；
New primers without attB * template→ CCR7)
4. Agarose Gel Electrophoresis

5. Purify the PCR products and detect the concentration of them.
6. PCR + Agarose Gel Electrophoresis: 50μl system
TA primers (CCR7) * new template
TA primers (CXCR5) * template

7. Vector 2: Combine CCR7/CXCR5 TA DNA and T3 vectors.
Transformation: Transfer vector 2 into trans5a bacteria and culture the modified bacteria for 16 hours.
8. Confirm whether we successfully transfer CXCR5 and CCR7 plasmids (TA primer) into the bacteria.

9. Amply the bacteria culture, store them, extract the plasmids(TA-CXCR5, TA-CCR7) from them and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
10. PCR：attB primers(CXCR5, CCR7) * plasmid templates, and Agarose Gel Electrophoresis 10μl
11. PCR: TA primers（CXCR1, CXCR3, CXCR4, CCR2, CCR5）* template，Agarose Gel Electrophoresis 10μl
Surprise: CCR2, CXCR3

12. PCR: attB primers (CXCR5, CCR7) * plasmid templates, and Agarose Gel Electrophoresis 50μl

13. PCR: TA primers（CXCR1, CXCR3, CXCR4, CCR2, CCR5）* template，Agarose Gel Electrophoresis 50μl

14. Purify the CXCR5, CCR7 DNA (both containing attB primer) and detect the concentration of them (TIANGEN kit)
15. Gateway: BP reaction (CXCR5, CCR7 DNA (both containing attB primer))
16. TA primers（CXCR1, CXCR3, CXCR4, CCR2）* template cDNA, Agarose Gel Electrophoresis 10μl

17. TA primers（CXCR3, CCR2）* template，Agarose Gel Electrophoresis 50μl

18. Continue Gateway from Experiment (Confirm whether we successfully transfer Entry clone: CXCR5-1064 vector and CCR7-1064 vector into the bacteria, respectively.)

19. Amply the modified bacteria culture
Incubate the bacteria culture for 16 hours at 37°C with vigorous agitation, monitoring the growth of the culture.
20. Continue Gateway (Preparation of Plasmid DNA (Entry clone: CXCR5-1064 vector and CCR7-1064 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
21. DNA sequence: Entry clone: CXCR5-1064 vector and CCR7-1064 vector
(CXCR5-1064 vector and CCR7-1064 vector pDown vector are finished.)
1. PCR: change annealing temperature (CXCR1, CXCR3, CXCR4, CCR2, CCR5 gene)
2. Agarose Gel Electrophoresis

3. PCR: new primers (CXCR4) * a new blood template, and Agarose Gel Electrophoresis 80μl

4. Purify the PCR product from step 3
5. Vector 2: Combine CXCR4 TA DNA and T3 vectors.
Transfer vector 2 into trans5a bacteria and culture the modified bacteria for 16 hours.
6. PCR: new primers (CXCR1, CXCR3, CCR2, CCR5 gene) * a new blood template2, and Agarose Gel Electrophoresis 10μl

7. PCR: new primers(CXCR1, CXCR3, CCR2, CCR5 gene) * a new blood template2, and Agarose Gel Electrophoresis 80μl

8. Confirm whether we successfully transfer CXCR4 plasmids (TA primer) into the bacteria and amply the bacteria culture and store them.

9. Purify the PCR products yesterday and detect the concentration of them. (CXCR1, CXCR3, CCR2, CCR5)
10. Vector 2: Combine CXCR1/CXCR3/CCR2/CCR5 TA DNA and T3 vectors.
11. Transfer vector 2 into trans5α and culture them for 16 hours.
12. Confirm whether we successfully transfer CXCR1/CXCR3/CCR2/CCR5 plasmids (TA primer) into the bacteria and amply the bacteria culture and store them.
13. Extract the plasmids (TA-CXCR1/ CXCR4/ CCR5) from them.
14. PCR：attB primers (CXCR1, CXCR4, CCR5) * plasmid templates, and Agarose Gel Electrophoresis 10μl

15. PCR：attB primers (CXCR1, CXCR4, CCR5) * plasmid templates, and Agarose Gel Electrophoresis 80μl

16. Purify PCR products
17. Gateway: BP reaction(CXCR1/ CXCR4/ CCR5-vector 1064)
18. Transfer plasmids (CXCR1/ CXCR4/ CCR5-vector 1064) into trans5α and culture the modified bacteria for 14 hours.
19. Pick a single bacterial colony again. Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
20. Continue Gateway (Confirm whether we successfully transfer Entry clone: CXCR1-1064 vector, CXCR4-1064 vector and CCR5-1064 vector into the bacteria, respectively.)

21. Amply the modified bacteria culture
Incubate the bacteria culture for 16 hours at 37°C with vigorous agitation, monitoring the growth of the culture.
22. Confirm whether we successfully transfer CCR2 plasmids (T3) into the bacteria and amply the bacteria culture and store them.

23. Preparation of Plasmid DNA (CCR2-T3 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
24. PCR: attB primers (CCR2) * CCR2-T3 vector and Agarose Gel Electrophoresis 100μl

25. Purify the PCR product and detect its concentration.
1. Continue Gateway (Preparation of Plasmid DNA (Entry clone: CXCR1-1064 vector, CXCR4-1064 vector and CCR5-1064 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
2. DNA sequence: Entry clone: CXCR1-1064 vector, CXCR4-1064 vector and CCR5-1064 vector.
3. Gateway: BP reaction (CCR2-vector 1064)
Transfer plasmids (CCR2-vector 1064) into trans5α and culture the modified bacteria for 14 hours.
4. Continue Gateway (Confirm whether we successfully transfer Entry clone: CCR2-1064 vector into the bacteria and culture them at 37°C for 14 hours.)

5. Explore CXCR3——PCR and Agarose Gel Electrophoresis
A new blood template * new primers (CXCR3) 10μl

6. PCR and Agarose Gel Electrophoresis
A new blood template * new primers (CXCR3) 100μl

7. Purify the PCR product from step 3 and detect its concentration.
Fail~~TOO LOW.
8. Preparation of Plasmid DNA (Entry clone: CCR2-1064 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
9. DNA sequence: Entry clone: CCR2-1064 vector.
10. Design point mutation of CXCR1, CXCR3, CXCR5, CCR2, CCR5
11. DNA sequence: dTomato, eGFP, luciferase-Ires-eGFP
1. Single point mutation for CXCR1, CXCR5, CCR2, CCR5
Agarose Gel Electrophoresis

2. Purify intermediate product of CXCR5/CCR5 and form mixture
3. Agarose Gel Electrophoresis

4.Repeat single point mutation of CXCR1 and CCR2.
5. Amply the plasmid backbone pSB1C3 and DNA sequence
6. Purify CXCR5 and CCR5 single-point mutation product and combine them with T3 vector, respectively.
7. Transfer CXCR5-T3 and CCR5-T3 vector into trans5α bacteria, respectively and culture them at 37°C for 14 hours.
8. Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.
9. Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR5 and CCR5 single-point mutation product)
10. PCR: dTomato, eGFP, IRES, luciferase.
11. Purify CXCR1 and CCR2 single-point mutation product and combine them with T3 vector, respectively.
12. Transfer CXCR1-T3 and CCR2-T3 vector into trans5α bacteria, respectively and culture them at 37°C for 14 hours.
13. Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.
14. Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR1 and CCR2 single-point mutation product)
15 Repeat single point mutation of CXCR1 and CCR2
RESULTS: fail to finish the point mutations of CXCR1 and CCR2
1 continue to conduct the point mutation of CXCR1 and CCR2
Results: NO. Now we change our method to conduct our point mutation of CXCR1 & CCR2
2 PCR: Single point mutation for CXCR1, Agarose Gel Electrophoresis

3 overlap of anterior fragment and posterior fragment (PCR)
Agarose Gel Electrophoresis

RESULTS: NO
4 Using another method to run the point mutation of CXCR1 (PCR to lengthen the posterior fragment to make an entire fragment)
5 PCR: the first time (adding 32bp to the posterior fragment)
Agarose Gel Electrophoresis

6 PCR: fragment IRES to dTomato
Agarose Gel Electrophoresis

RESULTS: NO
1 PCR: the second time (adding 34bp to the posterior+32bp fragment)
Agarose Gel Electrophoresis

2 Purify CXCR1 single-point mutation product and combine it with T3 vector
3 Transfer CXCR1-T3 vector into trans5α bacteria and culture it at 37°C for 14 hours.
4 Pick up a single clone of bacteria from step3 and confirm whether we successfully transfer them into bacteria.

5 Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR1 single-point mutation product)
So far, we have conduct all our chemokine receptors except for CXCR3, and finish the point mutation of CXCR1, CXCR5, CCR5 (CXCR4, CCR7 do not have to conduct the point mutation experiment.)
6 Conduct the Gateway LR reactions (EF-1α-CXCR4-IRES-eGFP)
7 Transfer plasmid (EF-1α-CXCR4-IRES-eGFP) into trans5α and culture the modified bacteria for 14 hours.
8 Pick a single bacterial colony from plates. And run a PCR to test the results

9 Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
10 Preparation of Plasmid DNA (expression vector: EF-1α-CXCR4-IRES-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
6 DNA sequence: expression vector: EF-1α-CXCR4-IRES-eGFP
Finish conduct the expression vector: EF-1α-CXCR4-IRES-eGFP
7 PCR：attB primers (CXCR5 point mutation) * plasmid templates from CXCR5 point mutation plasmid, and Agarose Gel Electrophoresis 80μl

8 Purify PCR products
9 Gateway: BP reaction (CXCR5 point mutation-vector 1064)
10 Transfer plasmids (CXCR5 point mutation-vector 1064) into trans5α and culture the modified bacteria for 14 hours.
11 Pick a single bacterial colony. And run a PCR and Agarose Gel Electrophoresis to test the results

12 Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
13 Preparation of Plasmid DNA (entry clone: CXCR5 point mutation -vector 1064) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
14 DNA sequence: CXCR5 point mutation-vector 1064
Finish conduct the CXCR5 point mutation-vector 1064
1 PCR: α-SMA promoter and run a Agarose Gel Electrophoresis to confirm the results

2 purify the PCR product
3 conduct Gateway: BP reaction——Pup α-SMA promoter
4 Transfer plasmids(Pup α-SMA promoter) into trans5α and culture the modified bacteria for 14 hours.
5 Pick a single bacterial colony. And run a PCR and Agarose Gel Electrophoresis to test the results

6 Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
7 Preparation of Plasmid DNA (entry clone: pup-α-SMA promoter) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
8 Gateway:LR reaction(α-SMA promoter-eGFP)
9 Transfer plasmid (α-SMA promoter-eGFP) into trans5α and culture the modified bacteria for 14 hours.
10 Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results

11 Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
12 Preparation of Plasmid DNA (expression vector: α-SMA promoter-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
13 DNA sequence: expression vector: α-SMA promoter-eGFP
Finish conduct the expression vector: α-SMA promoter-eGFP
14 PCR: CXCR5 point mutation+T2A (anterior fragment, short for R1); T2A (posterior fragment, short for F1) + luciferase
15 run an Agarose Gel Electrophoresis to test the results

16 Purify CXCR5-R1 and F1-luciferase product and combine them with T3 vector, respectively.
17 Transfer CXCR5-R1-T3 and F1-luciferase-T3 vector into trans5α bacteria, respectively and culture them at 37°C for 14 hours.
18 Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.

19 Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR5-R1 & F1-luciferase)
20 PCR the second time: CXCR5 point mutation + T2A (anterior fragment 2, short for R2) and T2A (posterior fragment 2, short for F2) + luciferase
21 run an Agarose Gel Electrophoresis to test the results

22 purify the PCR products of CXCR5-R2 & F2 luciferase
23 PCR: overlap of CXCR5-R2 & F2-luciferase ( short for CXCR5-luciferase attB)
24 run an Agarose Gel Electrophoresis to test the results

25 purify the PCR products of CXCR5-Luciferase attB
26 Gateway: BP reaction (CXCR5-Luciferase-vector 1064)
27 Transfer plasmids (CXCR5-Luciferase-vector 1064) into trans5α and culture the modified bacteria for 14 hours.
28 Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results

29 Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
30 Preparation of Plasmid DNA (entry clone: CXCR5-luciferase -vector 1064) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
31 DNA sequence: CXCR5-luciferase -vector 1064
Finish conduct the CXCR5 point mutation -vector 1064
32 Gateway: LR reaction (EF-1α-CXCR5-luciferase-IRES-eGFP)
33 Transfer plasmid (EF-1α-CXCR5-luciferase-IRES-eGFP) into trans5α and culture the modified bacteria for 14 hours.
34 Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results

35 Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
36 Preparation of Plasmid DNA (expression vector: EF-1α-CXCR5-luciferase-IRES-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
37 DNA sequence: expression vector: EF-1α-CXCR5-luciferase-IRES-eGFP
Finish conduct the expression vector: EF-1α-CXCR5-luciferase-IRES-eGFP
1 PCR: CXCR4 +T2A (anterior fragment, short for R1);
2 Run an Agarose Gel Electrophoresis to test the results

3 Purify CXCR4-R1 product and combine with T3 vector.
4 Transfer CXCR4-R1-T3 into trans5α bacteria, respectively and culture it at 37℃ for 14 hours.
5 Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.

6 Culture the single clone bacteria at 37℃ for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR4-R1)
7 PCR the second time: CXCR4+T2A (anterior fragment 2, short for R2)
8 run an Agarose Gel Electrophoresis to test the results

9 purify the PCR products of CXCR4-R2
10 PCR: overlap of CXCR4-R2 & F2-luciferase (short for CXCR4-luciferase attB)
11 run an Agarose Gel Electrophoresis to test the results

12 purify the PCR products of CXCR4-Luciferase attB
13 Gateway: BP reaction (CXCR4-Luciferase-vector 1064)
14 Transfer plasmids (CXCR4-Luciferase-vector 1064) into trans5α and culture the modified bacteria for 14 hours.
15 Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results

16 Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
17 Preparation of Plasmid DNA (entry clone: CXCR4-luciferase -vector 1064) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
18 DNA sequence: CXCR4-luciferase-vector 1064
Finish conduct the CXCR4 point mutation-vector 1064
19 Gateway: LR reaction (EF1-a-CXCR4-luciferase-IRES-eGFP)
20 Transfer plasmid (EF1-a-CXCR4-luciferase-IRES-eGFP) into trans5α and culture the modified bacteria for 14 hours.
21 Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results

22 Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
23 Preparation of Plasmid DNA (expression vector: EF1-a-CXCR4-luciferase-IRES-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
24 DNA sequence: expression vector: EF1-a-CXCR4-luciferase-IRES-eGFP
Finish conduct the expression vector: EF1-a-CXCR4-luciferase-IRES-eGFP
1 PCR: Overlap method to conduct the point mutation of luciferase + dTomato (point mutation) + hFTH
2 Agarose Gel Electrophoresis to test the results

3 purify the PCR products of luciferase+dTomato (anterior fragment) and dTomato (posterior fragment)+hFTH
4 PCR: overlap of luciferase+dTomato (anterior fragment) and dTomato (posterior fragment)+hFTH
5 Agarose Gel Electrophoresis to test the results

6 PCR: Overlap method to conduct the point mutation of luciferase+dTomato+hFTH(point mutation)
7 Agarose Gel Electrophoresis to test the results

8 purify the PCR products of dTomato (posterior fragment)+hFTH(anterior fragment)+hFTH (posterior fragment)
9 PCR: overlap of dTomato (posterior fragment)+hFTH(anterior fragment)+hFTH (posterior fragment)
10 Agarose Gel Electrophoresis to test the results

11 purify the PCR products of dTomato+hFTH (point mutaion)
12 PCR: overlap of luciferase+dTomato (anterior fragment) & dTomato (posterior fragment)+hFTH(anterior fragment)+hFTH (posterior fragment)
13 Agarose Gel Electrophoresis to test the results

14 purify the PCR products of Luciferase-dTomato(point mutation)-hFTH(pointi mutation)
15 PCR：IRES fragment
16 Agarose Gel Electrophoresis to test the results

17 purify the PCR product of IRES
18 PCR: Overlap of IRES & luciferase+dTomato+hFTH( including the attB)
19 Agarose Gel Electrophoresis to test the results

20 purify the product of IRES- luciferase-dTomato-hFTH( including the attB)
21 Gateway: BP reaction(IRES-luciferase-dTomato-hFTH-vector ptail)
22 Transfer plasmids (IRES-luciferase-dTomato-hFTH-vector ptail) into trans5α and culture the modified bacteria for 14 hours.
23 Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results

24 Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
25 Preparation of Plasmid DNA (entry clone: IRES-luciferase-dTomato-hFTH-vector ptail) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
26 DNA sequence: IRES-luciferase-dTomato-hFTH-vector ptail
Finish conduct the entry clone: IRES-luciferase-dTomato-hFTH-vector ptail
27 Gateway: LR reaction (EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH)
28 Transfer plasmid (EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH) into trans5α and culture the modified bacteria for 14 hours.
29 Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results

30 Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.
31 Preparation of Plasmid DNA (expression vector: EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)
32 DNA sequence: expression vector: EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH
Finish conduct the expression vector: EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH
Cell Experiment
293FT cells culture
Virus transduction to 293FT cells
Virus transduction to hMSC and culture
293FT cells culture
Virus transduction to 293FT cells
Virus transduction to hMSC and culture
In Vitro Confirmation
1. qPCR: Expression of chemokine receptors and CD markers on the surface of hMSCs

2.Confirm whether MSCs modified express fluorescent protein.

hMSC (CXCR4: eGFP) hMSC (CXCR5: eGFP) hMSC (CXCR4: dTomato)
1.Confirm whether MSCs remain their characteristics after modified.
2.Transwell: Confirm the function of hMSCs modified by CXCR4/CXCR5

3. qPCR and western blot: Confirm whether CXCR4/CXCR5 express on the surface of hMSCs
1.Switch confirmation
qPCR: Expression of alpha-SMA on hMSCs before and after adding TGF-β

2. hMSCs modified by switch plasmid express eGFP after adding TGF-β
Animal Experiment
IBD
1. Carefully shaved a 1cm×1cm filed on the back between the shoulders of the mouse using an electric razor.
2. While holding the mouse with one hand, smeared the shaved backs with presensitization solution (150μL/20g), which is the mixture of acetone, olive oil and TNBS in 16:4:5 scale.
3. Control mice are treated with presensitization solution without TNBS.
4. All the mice were in good condition.
DTH
1. Carefully shaved a 1cm×1cm filed on the back between the shoulders of the mouse using an electric razor.
2. While holding the mouse with one hand, smeared shaved backs with 150μL 0.5% DNFB solution in acetone/olive oil (4:1).
3. Control mice are treated with identical amount of acetone/olive oil (4:1).
4. All the mice were in good condition.
IBD
Absolute diet is applied to the mice apart from the water.
Anesthetize the mouse by intraperitoneal injection of 4% chloral hydrate (150μL/20g).
Insert the catheter into the colon 4cm proximal to the anus and slowly administer 150μL/20g TNBS/alcohol solution into the colon. Keep the mice with hand down for 60s before returning to the cage.
Concerning the damaged colon, we replace normal mice food with jelly to lessen the pain ensuing from sensitization.
In the evening, 3 mice are found dead, 2 mice are weak and have less activity, while the others are in good condition.
IBD
Weak mice are dead finally.
We weight the mice left. The weight change indicates that 18 of them are modeled successfully. So, we inject 200 μL MSCs in dose of 1*106 into 9 mice from caudal vein.
DTH
Challenge right ears with 20μL 0.3% DNFB solution. Two hours after sensitization, inject 200 μL MSCs in dose of 1*106 to 16 mice and PBS of identical amount to another 8 mice of control groups from caudal veins respectively.
IBD
All the animals are sacrificed by cervical dislocation. Then we take the colon tissue and measured the length .The colon tissue are kept in PFA or Trizol in EP tube for HE staining or q-PCR. The colon length, DAI score and the concentration of the cytokines will be performed using SPSS.
DTH
All the animals are sacrificed by cervical dislocation. Then we take the ear tissue and measured the thickness .The ear tissues are kept in PFA or Trizol in EP tube for HE staining or q-PCR. The ear thickness and the concentration of the cytokines will be performed using SPSS.
Start animal experiments for mathematical modeling and cooperation project with Team ShanghaiTechChina_B
Day 1, carefully shave a 1cm×1cm filed on the back between the shoulders of the mouse using an electric razor.
While holding the mouse with one hand, smear the shaved abdominal skin with presensitization solution (150μL/20g), which is the mixture of acetone, olive oil and TNBS in 16:4:5 scale.
Control mice are treated with presensitization solution without TNBS.
All the mice are in good condition.
Leave the mice until day 7.
On day 7, absolute diet is applied to the mice apart from the water.
On day 8, anesthetize the mouse by intraperitoneal injection of 4% chloral hydrate (150μL/20g). Insert the catheter into the colon 4cm proximal to the anus and slowly administer 150μL/20g TNBS/alcohol solution into the colon. Keep the mice with hand down for 60s before returning to the cage. Concerning the damaged colon, we take the jelly for the mice to lessen the pain after modelling.
On Day 11, dissolve 5 μg of recombinational EGF in 25 ml saline. Insert 150ug/20g EGF solution into the colon for 6 mice. Sacrifice the narcotic mice with EGF on Day 13.Take the large intestine tissue and measured the length. Keep the large intestine in PFA or Trizol in EP tube for qPCR or HE staining. The colon length, DAI score and the concentration of the cytokines will be performed using SPSS.

The mice left were injected 200 μL MSCs in dose of 1*106 into from caudal vein. At 5min, 30 min, 1h, 2h, 4h, 8h, 16h, 24h, 32h, 40h, 48h, 72h after injection, 1 narcotic mouse is taken living image by IVIS spectrum. After that, the mouse are sacrificed and colons are taken and measured for their length. The colons are kept in PFA or Trizol in EP tube for qPCR or HE staining.
Submission
Using the DNA carrier provided by iGEM, and the DNA to submit, digest and ligate them. But we failed. After analyzing, the reasons we suspect are as followed:
1.The concentration of the DNA is too low to make it,
2.The carrier provided by iGEM is useless because the linear carrier has low working efficiency.
As a result, we are going to have some meetings to solve it.
3.Using the part distribution to get the carrier annular carrier by Enzyme digestion, and put the first group of gene into the carrier by ligament.
Successful!
As a result, we finish the first group of submission, including two genes: CCR7 and CXCR4.
1.We started working the second group, including CXCR1, CCR5, Luciferease-IRES-eGFP, Luciferase-dTomato-hFTH, CXCR4-IRES-eGFP.
2.After four days’work, we finally finished the second group and put them all into a 96-well format.
1.Using the part distribution to get another carrier annular carrier by Enzyme digestion, and put the third group of genes into the carrier by ligament.
The third group includes CXCR5-Luciferease-IRES-eGFP, CXCR4-Luciferease-IRES-eGFP, CXCR4-Luciferase-dTomato-hFTH
2.After four days’work, we finally finished the third group and put them all into another 96-well format.

+

-

@@ Line 203: / Line 203: @@
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-             vertical-align: text-top;
+             vertical-align: top;
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          table.content-table tr td.center{
@@ Line 221: / Line 221: @@
          table.content-table tr.blank{
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+        }
+        table.content-table tr td.left a{
+            color:#333;
+            font-size: 18px;
+            line-height:2em;
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+        table.content-table tr td.left a:hover, table.content-table tr td.left a:active, table.content-table tr td.left a:focus{
+            color: #30CB8A;
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-             width: 960px;
+             width: 740px;
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@@ Line 233: / Line 241: @@
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-             width: 960px;
+             width: 740px;
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@@ Line 240: / Line 248: @@
              list-style: none;
              float: left;
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+             height: 40px;
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-             line-height: 38px;
+             line-height: 36px;
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@@ Line 255: / Line 264: @@
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+            position: relative;
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+             height: 1300px;
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              float: left;
@@ Line 445: / Line 458: @@
 <div class="jumbotron" id="content">
      <div class="my-content">
+        <br/>
          <table class="content-table">
              <tr>
-                 <td class="left">
+                 <td class=left>
-                     <ul class="nav nav-pills nav-stacked" role="tablist">
+                     <a href="#Story_Began"  class="tline-trigger">Story Began</a><br/>
-                        <li role="presentation" class="active"><a href="#preparation" aria-controls="preparation" role="tab" data-toggle="pill">Preparation</a></li>
+                    <a href="#Molecular_Cloning"  class="tline-trigger">Molecular Cloning</a><br/>
-                        <li role="presentation"><a href="#molecular" aria-controls="molecular" role="tab" data-toggle="pill">Molecular Cloning</a></li>
+                    <a href="#Cell_Experiment"  class="tline-trigger">Cell Experiment</a><br/>
-                        <li role="presentation"><a href="#cell" aria-controls="cell" role="tab" data-toggle="pill">Cell Experiment</a></li>
+                    <a href="#In_Vitro_Confirmation"  class="tline-trigger">In Vitro Confirmation</a><br/>
-                        <li role="presentation"><a href="#vitro" aria-controls="vitro" role="tab" data-toggle="pill">In Vitro Confirmation</a></li>
+                    <a href="#Animal_Experiment"  class="tline-trigger">Animal Experiment</a><br/>
-                        <li role="presentation"><a href="#animal" aria-controls="animal" role="tab" data-toggle="pill">Animal Experiment</a></li>
+                    <a href="#Submission"  class="tline-trigger">Submission</a><br/>
-                        <li role="presentation"><a href="#submission" aria-controls="submission" role="tab" data-toggle="pill">Submission</a></li>
-                    </ul>
                  </td>
-                <td class="padding"></td>
                  <td class="right">
-                     <div class="tab-content">
+                     <div id="timeline">
-                         <div role="tabpanel" class="tab-pane active" id="preparation">
+                         <style>
-                             <b>Preparation</b><br/>
+                             #issues p {
+                                width: 80%;
+                                margin: 0 auto;
+                            }
+                        </style>
-                        </div>
-                        <div role="tabpanel" class="tab-pane" id="molecular">
-                            <b>Molecular Cloning</b><br/>
-                         </div>
+                         <ul id="dates" style="width: 3030px; margin-left: -950px;">
-                        <div role="tabpanel" class="tab-pane" id="cell">
+                             <!--01Preparation-->
-                             <b>Cell Experiment</b><br/>
+                            <li><a href="#Story_Began" class="selected">Story Began</a></li>
-                        </div>
+                            <li><a href="#2015.11">2015.11</a></li>
-                        <div role="tabpanel" class="tab-pane" id="vitro">
+                             <li><a href="#2015.12">2015.12</a></li>
-                             <b>In Vitro Confirmation</b><br/>
+                            <li><a href="#2016.01">2016.01</a></li>
-                        </div>
+                            <li><a href="#2016.01~02">2016.01~02</a></li>
-                        <div role="tabpanel" class="tab-pane" id="animal">
+                            <li><a href="#2016.02.22">2016.02.22</a></li>
-                             <b>Animal Experiment</b><br/>
+                             <li><a href="#2016.02~03">2016.02~03</a></li>
-                        </div>
+                            <li><a href="#2016.03.04">2016.03.04</a></li>
-                        <div role="tabpanel" class="tab-pane" id="submission">
+                            <li><a href="#2016.03.11">2016.03.11</a></li>
-                             <b>Submission</b><br/>
+                            <li><a href="#2016.03.15">2016.03.15</a></li>
-                        </div>
+                             <li><a href="#2016.04~05">2016.04~05</a></li>
+                            <li><a href="#2016.06.05">2016.06.05</a></li>
-                    </div>
+                            <!--02Molecular Cloning-->
-                </td>
+                            <li><a href="#Molecular_Cloning">Molecular Cloning</a></li>
-            </tr>
+                            <li><a href="#2016.06.05-12">2016.06.05-12</a></li>
-        </table>
+                            <li><a href="#2016.06.13-19">2016.06.13-19</a></li>
-        <br/>
+                            <li><a href="#2016.06.20-26">2016.06.20-26</a></li>
-        <Br/>
+                            <li><a href="#2016.06.27-07.03">2016.06.27-07.03</a></li>
-        <div id="timeline">
+                            <li><a href="#2016.07.04-10">2016.07.04-10</a></li>
-            <style>
+                            <li><a href="#2016.07.11-17">2016.07.11-17</a></li>
-                #issues p {
+                            <li><a href="#2016.07.18-24">2016.07.18-24</a></li>
-                    width: 80%;
+                            <li><a href="#2016.07.25-31">2016.07.25-31</a></li>
-                    margin: 0 auto;
+                            <li><a href="#2016.08.01-07">2016.08.01-07</a></li>
-                }
+                            <li><a href="#2016.08.08-14">2016.08.08-14</a></li>
-            </style>
+                            <!--03Cell Experiment-->
+                            <li><a href="#Cell_Experiment">Cell Experiment</a></li>
+                            <li><a href="#2016.08.15-21">2016.08.15-21</a></li>
+                            <li><a href="#2016.08.22-28">2016.08.22-28</a></li>
+                            <li><a href="#2016.08.29-09.04">2016.08.29-09.04</a></li>
+                            <!--04In Vitro Confirmation-->
+                            <li><a href="#In_Vitro_Confirmation">In Vitro Confirmation</a></li>
+                            <li><a href="#2016.09.05-11">2016.09.05-11</a></li>
+                            <li><a href="#2016.09.12-18">2016.09.12-18</a></li>
+                            <li><a href="#2016.09.19-25">2016.09.19-25</a></li>
+                            <!--05Animal Experiment-->
+                            <li><a href="#Animal_Experiment">Animal Experiment</a></li>
+                            <li><a href="#2016.09.05">2016.09.05</a></li>
+                            <li><a href="#2016.09.07">2016.09.07</a></li>
+                            <li><a href="#2016.09.11">2016.09.11</a></li>
+                            <li><a href="#2016.09.12">2016.09.12</a></li>
+                            <li><a href="#2016.09.13">2016.09.13</a></li>
+                            <li><a href="#2016.09.14">2016.09.14</a></li>
+                            <li><a href="#2016.09.15-22">2016.09.15-22</a></li>
+                            <li><a href="#2016.09.16-21">2016.09.16-21</a></li>
+                            <li><a href="#2016.09.17">2016.09.17</a></li>
+                            <li><a href="#2016.09.17-24">2016.09.17-24</a></li>
+                            <li><a href="#2016.09.25">2016.09.25</a></li>
+                            <li><a href="#2016.09.28-10.05">2016.09.28-10.05</a></li>
-            <ul id="dates" style="width: 3030px; margin-left: -950px;">
+                            <!--06Submission-->
-                <li><a href="#2015-04-16" class="">2015-04-16</a></li>
+                            <li><a href="#Submission">Submission</a></li>
-                <li><a href="#2015-04-25" class="">2015-04-25</a></li>
+                            <li><a href="#2016.09.26-29">2016.09.26-29</a></li>
-                <li><a href="#2015-05-01" class="">2015-05-01</a></li>
+                            <li><a href="#2016.10.04-09">2016.10.04-09</a></li>
-                <li><a href="#2015-05-08" class="">2015-05-08</a></li>
+                            <li><a href="#2016.10.09-15">2016.10.09-15</a></li>
-                <li><a href="#2015-05-12" class="">2015-05-12</a></li>
-                <li><a href="#2015-05-14" class="">2015-05-14</a></li>
-                <li><a href="#2015-05-26" class="">2015-05-26</a></li>
-                <li><a href="#2015-06-01" class="">2015-06-01</a></li>
-                <li><a href="#2015-06-07" class="">2015-06-07</a></li>
-                <li><a href="#2015-06-12" class="">2015-06-12</a></li>
-                <li><a href="#2015-06-18" class="">2015-06-18</a></li>
-                <li><a href="#2015-06-25" class="">2015-06-25</a></li>
-                <li><a href="#2015-07-01" class="">2015-07-01</a></li>
-                <li><a href="#2015-07-10" class="selected">2015-07-10</a></li>
-                <li><a href="#2015-07-15">2015-07-15</a></li>
-                <li><a href="#2015-07-18">2015-07-18</a></li>
-                <li><a href="#2015-07-23">2015-07-23</a></li>
-                <li><a href="#2015-07-24">2015-07-24</a></li>
-                <li><a href="#2015-07-29">2015-07-29</a></li>
-                <li><a href="#2015-08-01">2015-08-01</a></li>
-                <li><a href="#2015-08-04">2015-08-04</a></li>
-                <li><a href="#2015-08-10">2015-08-10</a></li>
-                <li><a href="#2015-08-17">2015-08-17</a></li>
-                <li><a href="#2015-08-25">2015-08-25</a></li>
-                <li><a href="#2015-08-28">2015-08-28</a></li>
-                <li><a href="#2015-09-01">2015-09-01</a></li>
-                <li><a href="#2015-09-06">2015-09-06</a></li>
-                <li><a href="#2015-09-10">2015-09-10</a></li>
-                <li><a href="#2015-09-12">2015-09-12</a></li>
-                <li><a href="#2015-09-14">2015-09-14</a></li>
-            </ul>
-            <ul id="issues" style="width: 24030px; margin-left: -10400px;">
+                        </ul>
-                <li id="2015-04-16" class="" style="opacity: 0.2;">
+                        <ul id="issues" style="width: 24030px; margin-left: -10400px;">
-                    <p>
+                            <!--01Preparation-->
-                        -culture the competence cell
+                            <li id="Story_Began" class="" style="opacity: 0.2;">
-                        <br>
+                                <p></br>
-                        -test the experiment kits
+                                    Since it is the first time for our school to participate in the iGEM competition ……
-                    </p>
+                                </p>
-                </li>
+                            </li>
+                            <li id="2015.11" class="" style="opacity: 0.2;">
+                                <p></br>
+                                    Having basic knowledge of iGEM, six kindred spirits gathered together and decided to participate in this competition. After deliberation, we started to prepare our team.
+                                </p>
+                            </li>
+                            <li id="2015.12" class="" style="opacity: 0.2;">
+                                <p></br>
+                                    Our team, named as SYSU-MEDICINE, was successfully built, comprising four groups for lab work, modeling, art design and human practice respectively.
+                                </p>
+                            </li>
+                            <li id="2016.01" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Assigned the tasks for winter vacation, including exploring feasible project theme, learned basic methods of literature search and summarized previous iGEM projects of the latest three years.
+                                </p>
+                            </li>
+                            <li id="2016.01~02" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Six internal meetings were held and had a meet up with SYSU-China, from which we learned the importance of having a round knowledge of the rules of iGEM competition.
+                                </p>
+                            </li>
+                            <li id="2016.02.22" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Started raising fund and lab resources.
+                                </p>
+                            </li>
+                            <li id="2016.02~03" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Got in touch with leadership of Zhongshan School of Medicine and several professors and finally gained official support of Zhongshan School of Medicine. Refined our project at the suggestion of professors.
+                                </p>
+                            </li>
+                            <li id="2016.03.04" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Final determination of supporting lab and fund resource.
+                                </p>
+                            </li>
+                            <li id="2016.03.11" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Determined our first PI, second PI and instructor, to whom we presented our primary project and had a further discussion.
+                                </p>
+                            </li>
+                            <li id="2016.03.15" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Signed up for iGEM.
+                                </p>
+                            </li>
+                            <li id="2016.04~05" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Internal meetings for routine progress report were held, in which we decided the main topic of our project: mesenchymal stem cells (MSCs). After related article readings and with the guidance of our instructor, we further enriched our project and detailed our experiment plans. On May 20th, our final design was born.
+                                </p>
+                            </li>
+                            <li id="2016.06.05" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Experiments begin.
+                                </p>
+                            </li>
-                 <li id="2015-04-25" class="" style="opacity: 0.2;">
+                            <!--02Molecular Cloning-->
-                    <p>
+                            <li id="Molecular_Cloning" class="" style="opacity: 0.2;">
-                        -Molecular cloning for Bba_J06602, BBa_B0012, Bba_K592024, Bba_I13453, Bba_B0010
+                                <p></br>
-                        <br>
+                                    Molecular Cloning
-                        -Tried to reverse pBAD(BBa_I13453)
+                                </p>
-                        <br>
+                            </li>
-                        -preparation of the target bio-brick
+                            <li id="2016.06.05-12" class="" style="opacity: 0.2;">
-                    </p>
+                                <p>
-                </li>
+                                    </br>
+. Extraction, Purification, and Analysis of total RNA from PBMCs (peripheral blood mononuclear cells)<br/>
+. RT-PCR——DNA : RNA hybridization<br/>
+. PCR, agarose gel electrophoresis, (primers without attB * cDNA; new primers with attB * cDNA)     10μl<br/>
+                                    <br/>
+bp marker  positive control                 100bp marker only<br/>
+. Extraction, Purification, and Analysis of total RNA from new PBMCs (peripheral blood mononuclear cells) and RT-PCR——DNA : RNA hybridization<br/>
+                                </p>
+                            </li>
+                            <li id="2016.06.13-19" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+.  TIANprep Mini Plasmid Kit (TIANGEN)<br/>
+.  Continue experiment yesterday.<br/>
+                                    PCR (new primers without attB * template cDNA; new primers without attB * template cDNA; CXCR5 primer * CXCR5 plasmid DNA), agarose gel electrophoresis     10μl<br/>
+                                    <br/>
+. PCR <br/>
+                                    (new primers without attB * new template cDNA→ CXCR5；<br/>
+                                    New primers without attB * template→ CCR7) <br/>
+. Agarose Gel Electrophoresis<br/>
+                                    <br/>
+. Purify the PCR products and detect the concentration of them.<br/>
+. PCR + Agarose Gel Electrophoresis:  50μl system<br/>
+                                    TA primers (CCR7) * new template<br/>
+                                    TA primers (CXCR5) * template<br/>
+                                    <br/>
+. Vector 2: Combine CCR7/CXCR5 TA DNA and T3 vectors.<br/>
+                                    Transformation: Transfer vector 2 into trans5a bacteria and culture the modified bacteria for 16 hours.<br/>
+. Confirm whether we successfully transfer CXCR5 and CCR7 plasmids (TA primer) into the bacteria. <br/>
+                                    <br/>
+. Amply the bacteria culture, store them, extract the plasmids(TA-CXCR5, TA-CCR7) from them and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+. PCR：attB primers(CXCR5, CCR7) * plasmid templates, and Agarose Gel Electrophoresis             10μl<br/>
+. PCR: TA primers（CXCR1, CXCR3, CXCR4, CCR2, CCR5）* template，Agarose Gel Electrophoresis                 10μl<br/>
+                                    Surprise: CCR2, CXCR3 <br/>
+                                    <br/>
+. PCR: attB primers (CXCR5, CCR7) * plasmid templates, and Agarose Gel Electrophoresis                                50μl<br/>
+                                    <br/>
+. PCR: TA primers（CXCR1, CXCR3, CXCR4, CCR2, CCR5）* template，Agarose Gel Electrophoresis                   50μl<br/>
+                                    <br/>
+. Purify the CXCR5, CCR7 DNA (both containing attB primer) and detect the concentration of them (TIANGEN kit)<br/>
+. Gateway: BP reaction (CXCR5, CCR7 DNA (both containing attB primer)) <br/>
+. TA primers（CXCR1, CXCR3, CXCR4, CCR2）* template cDNA, Agarose Gel Electrophoresis                      10μl<br/>
+                                    <br/>
+. TA primers（CXCR3, CCR2）* template，Agarose Gel Electrophoresis                      50μl<br/>
+                                    <br/>
+. Continue Gateway from Experiment (Confirm whether we successfully transfer Entry clone: CXCR5-1064 vector and CCR7-1064 vector into the bacteria, respectively.)<br/>
+                                    <br/>
+. Amply the modified bacteria culture<br/>
+                                    Incubate the bacteria culture for 16 hours at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+. Continue Gateway (Preparation of Plasmid DNA (Entry clone: CXCR5-1064 vector and CCR7-1064 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+. DNA sequence: Entry clone: CXCR5-1064 vector and CCR7-1064 vector<br/>
+                                    (CXCR5-1064 vector and CCR7-1064 vector pDown vector are finished.)<br/>
+                                </p>
+                            </li>
+                            <li id="2016.06.20-26" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+. PCR: change annealing temperature (CXCR1, CXCR3, CXCR4, CCR2, CCR5 gene)<br/>
+. Agarose Gel Electrophoresis<br/>
+                                    <br/>
+. PCR: new primers (CXCR4) * a new blood template, and Agarose Gel Electrophoresis            80μl          <br/>
+                                    <br/>
+. Purify the PCR product from step 3<br/>
+. Vector 2: Combine CXCR4 TA DNA and T3 vectors.<br/>
+                                    Transfer vector 2 into trans5a bacteria and culture the modified bacteria for 16 hours.<br/>
+. PCR: new primers (CXCR1, CXCR3, CCR2, CCR5 gene) * a new blood template2, and Agarose Gel Electrophoresis            10μl          <br/>
+                                    <br/>
+. PCR: new primers(CXCR1, CXCR3, CCR2, CCR5 gene) * a new blood template2, and Agarose Gel Electrophoresis            80μl          <br/>
+                                    <br/>
+                                    <br/>
+. Confirm whether we successfully transfer CXCR4 plasmids (TA primer) into the bacteria and amply the bacteria culture and store them.<br/>
+                                    <br/>
+. Purify the PCR products yesterday and detect the concentration of them. (CXCR1, CXCR3, CCR2, CCR5)<br/>
+. Vector 2: Combine CXCR1/CXCR3/CCR2/CCR5 TA DNA and T3 vectors.<br/>
+. Transfer vector 2 into trans5α and culture them for 16 hours.<br/>
+. Confirm whether we successfully transfer CXCR1/CXCR3/CCR2/CCR5 plasmids (TA primer) into the bacteria and amply the bacteria culture and store them.  <br/>
+. Extract the plasmids (TA-CXCR1/ CXCR4/ CCR5) from them.<br/>
+. PCR：attB primers (CXCR1, CXCR4, CCR5) * plasmid templates, and Agarose Gel Electrophoresis             10μl<br/>
+                                    <br/>
+. PCR：attB primers (CXCR1, CXCR4, CCR5) * plasmid templates, and Agarose Gel Electrophoresis             80μl<br/>
+                                    <br/>
+. Purify PCR products<br/>
+. Gateway: BP reaction(CXCR1/ CXCR4/ CCR5-vector 1064)<br/>
+. Transfer plasmids (CXCR1/ CXCR4/ CCR5-vector 1064) into trans5α and culture the modified bacteria for 14 hours.<br/>
+. Pick a single bacterial colony again. Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+. Continue Gateway (Confirm whether we successfully transfer Entry clone: CXCR1-1064 vector, CXCR4-1064 vector and CCR5-1064 vector into the bacteria, respectively.)<br/>
+                                    <br/>
+                                    <br/>
+. Amply the modified bacteria culture<br/>
+                                    Incubate the bacteria culture for 16 hours at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+. Confirm whether we successfully transfer CCR2 plasmids (T3) into the bacteria and amply the bacteria culture and store them.<br/>
+                                    <br/>
+. Preparation of Plasmid DNA (CCR2-T3 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+. PCR: attB primers (CCR2) * CCR2-T3 vector and Agarose Gel Electrophoresis    100μl   <br/>
+                                    <br/>
+. Purify the PCR product and detect its concentration.<br/>
+                                    <br/>
+                                </p>
+                            </li>
+                            <li id="2016.06.27-07.03" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+. Continue Gateway (Preparation of Plasmid DNA (Entry clone: CXCR1-1064 vector, CXCR4-1064 vector and CCR5-1064 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+                                    <b>2.</b> DNA sequence: Entry clone: CXCR1-1064 vector, CXCR4-1064 vector and CCR5-1064 vector.<br/>
+. Gateway: BP reaction (CCR2-vector 1064)<br/>
+                                    Transfer plasmids (CCR2-vector 1064) into trans5α and culture the modified bacteria for 14 hours.<br/>
+. Continue Gateway (Confirm whether we successfully transfer Entry clone: CCR2-1064 vector into the bacteria and culture them at 37°C for 14 hours.)<br/>
+                                    <br/>
+. Explore CXCR3——PCR and Agarose Gel Electrophoresis       <br/>
+                                    A new blood template * new primers (CXCR3)          10μl<br/>
+                                    <br/>
+                                    <br/>
+                                    <br/>
+                                    <br/>
+                                    <br/>
+                                    <br/>
+                                    <br/>
+. PCR and Agarose Gel Electrophoresis       <br/>
+                                    A new blood template * new primers (CXCR3)        100μl<br/>
+                                    <br/>
+. Purify the PCR product from step 3 and detect its concentration.<br/>
+                                    Fail~~TOO LOW.<br/>
+. Preparation of Plasmid DNA (Entry clone: CCR2-1064 vector) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+. DNA sequence: Entry clone: CCR2-1064 vector.<br/>
+. Design point mutation of CXCR1, CXCR3, CXCR5, CCR2, CCR5 <br/>
+. DNA sequence: dTomato, eGFP, luciferase-Ires-eGFP<br/>
+                                </p>
+                            </li>
+                            <li id="2016.07.04-10" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+. Single point mutation for CXCR1, CXCR5, CCR2, CCR5<br/>
+                                    Agarose Gel Electrophoresis<br/>
+                                    <br/>
+. Purify intermediate product of CXCR5/CCR5 and form mixture<br/>
+. Agarose Gel Electrophoresis<br/>
+                                    <br/>
+.Repeat single point mutation of CXCR1 and CCR2. <br/>
+. Amply the plasmid backbone pSB1C3 and DNA sequence<br/>
+. Purify CXCR5 and CCR5 single-point mutation product and combine them with T3 vector, respectively.<br/>
+. Transfer CXCR5-T3 and CCR5-T3 vector into trans5α bacteria, respectively and culture them at 37°C for 14 hours.<br/>
+. Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.<br/>
+. Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR5 and CCR5 single-point mutation product)<br/>
+. PCR: dTomato, eGFP, IRES, luciferase.<br/>
+. Purify CXCR1 and CCR2 single-point mutation product and combine them with T3 vector, respectively.<br/>
+. Transfer CXCR1-T3 and CCR2-T3 vector into trans5α bacteria, respectively and culture them at 37°C for 14 hours.<br/>
+. Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.<br/>
+. Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR1 and CCR2 single-point mutation product)<br/>
+   Repeat single point mutation of CXCR1 and CCR2<br/>
+                                    RESULTS: fail to finish the point mutations of CXCR1 and CCR2<br/>
+                                </p>
+                            </li>
+                            <li id="2016.07.11-17" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+    continue to conduct the point mutation of CXCR1 and CCR2<br/>
+                                    Results: NO. Now we change our method to conduct our point mutation of CXCR1 & CCR2<br/>
+    PCR: Single point mutation for CXCR1, Agarose Gel Electrophoresis<br/>
+                                    <br/>
+    overlap of anterior fragment and posterior fragment (PCR) <br/>
+                                    Agarose Gel Electrophoresis<br/>
+                                    <br/>
+                                    RESULTS: NO<br/>
+   Using another method to run the point mutation of CXCR1 (PCR to lengthen the posterior fragment to make an entire fragment)<br/>
+   PCR: the first time (adding 32bp to the posterior fragment)<br/>
+                                    Agarose Gel Electrophoresis<br/>
+                                    <br/>
+    PCR: fragment IRES to dTomato<br/>
+                                    Agarose Gel Electrophoresis<br/>
+                                    <br/>
+                                    RESULTS: NO<br/>
+                                </p>
+                            </li>
+                            <li id="2016.07.18-24" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+   PCR: the second time (adding 34bp to the posterior+32bp fragment)<br/>
+                                    Agarose Gel Electrophoresis<br/>
+                                    <br/>
+    Purify CXCR1 single-point mutation product and combine it with T3 vector<br/>
+    Transfer CXCR1-T3 vector into trans5α bacteria and culture it at 37°C for 14 hours.<br/>
+   Pick up a single clone of bacteria from step3 and confirm whether we successfully transfer them into bacteria.<br/>
+                                    <br/>
+   Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR1 single-point mutation product) <br/>
+                                    So far, we have conduct all our chemokine receptors except for CXCR3, and finish the point mutation of CXCR1, CXCR5, CCR5 (CXCR4, CCR7 do not have to conduct the point mutation experiment.)<br/>
+    Conduct the Gateway LR reactions (EF-1α-CXCR4-IRES-eGFP)<br/>
+    Transfer plasmid (EF-1α-CXCR4-IRES-eGFP) into trans5α and culture the modified bacteria for 14 hours.<br/>
+    Pick a single bacterial colony from plates. And run a PCR to test the results<br/>
+                                    <br/>
+    Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+    Preparation of Plasmid DNA (expression vector: EF-1α-CXCR4-IRES-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: expression vector: EF-1α-CXCR4-IRES-eGFP<br/>
+                                    Finish conduct the expression vector: EF-1α-CXCR4-IRES-eGFP<br/>
+   PCR：attB primers (CXCR5 point mutation) * plasmid templates from CXCR5 point mutation plasmid, and Agarose Gel Electrophoresis             80μl<br/>
+                                    <br/>
+   Purify PCR products<br/>
+   Gateway: BP reaction (CXCR5 point mutation-vector 1064)<br/>
+   Transfer plasmids (CXCR5 point mutation-vector 1064) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+    Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+    Preparation of Plasmid DNA (entry clone: CXCR5 point mutation -vector 1064) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: CXCR5 point mutation-vector 1064<br/>
+                                    Finish conduct the CXCR5 point mutation-vector 1064<br/>
+                                </p>
+                            </li>
+                            <li id="2016.07.25-31" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+  PCR: α-SMA promoter and run a Agarose Gel Electrophoresis to confirm the results<br/>
+                                    <br/>
+   purify the PCR product <br/>
+   conduct Gateway: BP reaction——Pup α-SMA promoter<br/>
+   Transfer plasmids(Pup α-SMA promoter) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+   Preparation of Plasmid DNA (entry clone: pup-α-SMA promoter) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   Gateway:LR reaction(α-SMA promoter-eGFP)<br/>
+   Transfer plasmid (α-SMA promoter-eGFP) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+   Preparation of Plasmid DNA (expression vector: α-SMA promoter-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: expression vector: α-SMA promoter-eGFP<br/>
+                                    Finish conduct the expression vector: α-SMA promoter-eGFP<br/>
+   PCR: CXCR5 point mutation+T2A (anterior fragment, short for R1); T2A (posterior fragment, short for F1) + luciferase<br/>
+   run an Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Purify CXCR5-R1 and F1-luciferase product and combine them with T3 vector, respectively.<br/>
+   Transfer CXCR5-R1-T3 and F1-luciferase-T3 vector into trans5α bacteria, respectively and culture them at 37°C for 14 hours.<br/>
+    Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.<br/>
+                                    <br/>
+    Culture the single clone bacteria at 37°C for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR5-R1 & F1-luciferase)<br/>
+    PCR the second time: CXCR5 point mutation + T2A (anterior fragment 2, short for R2) and T2A (posterior fragment 2, short for F2) + luciferase <br/>
+    run an Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   purify the PCR products of CXCR5-R2 & F2 luciferase<br/>
+   PCR: overlap of CXCR5-R2 & F2-luciferase ( short for CXCR5-luciferase attB)<br/>
+   run an Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   purify the PCR products of CXCR5-Luciferase attB<br/>
+   Gateway: BP reaction (CXCR5-Luciferase-vector 1064)<br/>
+   Transfer plasmids (CXCR5-Luciferase-vector 1064) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+   Preparation of Plasmid DNA (entry clone: CXCR5-luciferase -vector 1064) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: CXCR5-luciferase -vector 1064<br/>
+                                    Finish conduct the CXCR5 point mutation -vector 1064<br/>
+   Gateway: LR reaction (EF-1α-CXCR5-luciferase-IRES-eGFP)<br/>
+   Transfer plasmid (EF-1α-CXCR5-luciferase-IRES-eGFP) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+   Preparation of Plasmid DNA (expression vector: EF-1α-CXCR5-luciferase-IRES-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: expression vector: EF-1α-CXCR5-luciferase-IRES-eGFP<br/>
+                                    Finish conduct the expression vector: EF-1α-CXCR5-luciferase-IRES-eGFP<br/>
+                                </p>
+                            </li>
+                            <li id="2016.08.01-07" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+   PCR: CXCR4 +T2A (anterior fragment, short for R1); <br/>
+   Run an Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Purify CXCR4-R1 product and combine with T3 vector. <br/>
+   Transfer CXCR4-R1-T3 into trans5α bacteria, respectively and culture it at 37℃ for 14 hours.<br/>
+    Pick up a single clone of bacteria and confirm whether we successfully transfer them into bacteria.<br/>
+                                    <br/>
+    Culture the single clone bacteria at 37℃ for 14 hours, extract the plasmids from them, detect the concentration of them, and DNA sequencing. (CXCR4-R1)<br/>
+    PCR the second time: CXCR4+T2A (anterior fragment 2, short for R2) <br/>
+    run an Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   purify the PCR products of CXCR4-R2 <br/>
+   PCR: overlap of CXCR4-R2 & F2-luciferase (short for CXCR4-luciferase attB)<br/>
+   run an Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   purify the PCR products of CXCR4-Luciferase attB<br/>
+   Gateway: BP reaction (CXCR4-Luciferase-vector 1064)<br/>
+   Transfer plasmids (CXCR4-Luciferase-vector 1064) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+   Preparation of Plasmid DNA (entry clone: CXCR4-luciferase -vector 1064) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: CXCR4-luciferase-vector 1064<br/>
+                                    Finish conduct the CXCR4 point mutation-vector 1064<br/>
+   Gateway: LR reaction (EF1-a-CXCR4-luciferase-IRES-eGFP)<br/>
+   Transfer plasmid (EF1-a-CXCR4-luciferase-IRES-eGFP) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+   Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+   Preparation of Plasmid DNA (expression vector: EF1-a-CXCR4-luciferase-IRES-eGFP) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: expression vector: EF1-a-CXCR4-luciferase-IRES-eGFP<br/>
+                                    Finish conduct the expression vector: EF1-a-CXCR4-luciferase-IRES-eGFP<br/>
+                                </p>
+                            </li>
+                            <li id="2016.08.08-14" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+   PCR: Overlap method to conduct the point mutation of luciferase + dTomato (point mutation) + hFTH<br/>
+   Agarose Gel Electrophoresis to test the results  <br/>
+                                    <br/>
+                                    <br/>
+   purify the PCR products of luciferase+dTomato (anterior fragment) and dTomato (posterior fragment)+hFTH<br/>
+    PCR: overlap of luciferase+dTomato (anterior fragment) and dTomato (posterior fragment)+hFTH<br/>
+    Agarose Gel Electrophoresis to test the results  <br/>
+                                    <br/>
+    PCR: Overlap method to conduct the point mutation of luciferase+dTomato+hFTH(point mutation)<br/>
+    Agarose Gel Electrophoresis to test the results  <br/>
+                                    <br/>
+    purify the PCR products of dTomato (posterior fragment)+hFTH(anterior fragment)+hFTH (posterior fragment)<br/>
+    PCR: overlap of dTomato (posterior fragment)+hFTH(anterior fragment)+hFTH (posterior fragment)<br/>
+    Agarose Gel Electrophoresis to test the results  <br/>
+                                    <br/>
+    purify the PCR products of dTomato+hFTH (point mutaion)<br/>
+    PCR: overlap of luciferase+dTomato (anterior fragment) & dTomato (posterior fragment)+hFTH(anterior fragment)+hFTH (posterior fragment)<br/>
+    Agarose Gel Electrophoresis to test the results  <br/>
+                                    <br/>
+   purify the PCR products of Luciferase-dTomato(point mutation)-hFTH(pointi mutation)<br/>
+    PCR：IRES fragment<br/>
+    Agarose Gel Electrophoresis to test the results   <br/>
+                                    <br/>
+    purify the PCR product of IRES<br/>
+    PCR: Overlap of IRES & luciferase+dTomato+hFTH( including the attB)<br/>
+    Agarose Gel Electrophoresis to test the results   <br/>
+                                    <br/>
+    purify the product of IRES- luciferase-dTomato-hFTH( including the attB)<br/>
+    Gateway: BP reaction(IRES-luciferase-dTomato-hFTH-vector ptail)<br/>
+    Transfer plasmids (IRES-luciferase-dTomato-hFTH-vector ptail) into trans5α and culture the modified bacteria for 14 hours.<br/>
+   Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+    Transfer the colony into medium (containing kana antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+    Preparation of Plasmid DNA (entry clone: IRES-luciferase-dTomato-hFTH-vector ptail) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: IRES-luciferase-dTomato-hFTH-vector ptail<br/>
+                                    Finish conduct the entry clone: IRES-luciferase-dTomato-hFTH-vector ptail<br/>
+    Gateway: LR reaction (EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH)<br/>
+    Transfer plasmid (EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH) into trans5α and culture the modified bacteria for 14 hours.<br/>
+    Pick a single bacterial colony from plates. And run a PCR and Agarose Gel Electrophoresis to test the results<br/>
+                                    <br/>
+    Transfer the colony into medium (containing Amp antibiotic). Incubate the culture for 12 hour at 37°C with vigorous agitation, monitoring the growth of the culture.<br/>
+    Preparation of Plasmid DNA (expression vector: EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH) by TIANprep Mini Plasmid Kit (TIANGEN) and estimate the concentration of the plasmid DNA by measuring the absorbance at 260 nm and 280nm of an aliquot of the final preparation. (Nanodrop machine)<br/>
+   DNA sequence: expression vector: EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH<br/>
+                                    Finish conduct the expression vector: EF1-a-CXCR4- IRES-luciferase-dTomato-hFTH<br/>
+                                </p>
+                            </li>
-                <li id="2015-05-01" class="" style="opacity: 0.2;">
+                            <!--03Cell Experiment-->
-                    <p>
+                            <li id="Cell_Experiment" class="" style="opacity: 0.2;">
-                        -Ligation: flpe-BFP, loxP-pBAD, B0010-loxP, eGFP-B0015, FRT-pBAD
+                                <p></br>
-                        <br>
+                                    Cell Experiment
-                        -Preservation of the plasmid pAUR135 in E.coli;
+                                </p>
-                        Transformation of the commercial plasmid pAUR135(from TAKARA, Item No.D3604);
+                            </li>
-                        select monoclonal colony and extract plasmid(pAUR135) from the culture.
+                            <li id="2016.08.15-21" class="" style="opacity: 0.2;">
-                        <br>
+                                <p>
-                        -Prepared Yeast Extract Peptone Dextrose(YPD) Medium
+                                    </br>
-                    </p>
+FT cells culture <br/>
-                </li>
+                                    Virus transduction to 293FT cells<br/>
-                <li id="2015-05-08" class="" style="opacity: 0.2;">
+                                </p>
-                    <p>
+                            </li>
-                        -Patch:
+                            <li id="2016.08.22-28" class="" style="opacity: 0.2;">
-                        strain: S.cerevisiae W303;
+                                <p>
-                        medium: YPD agar
+                                    </br>
-                    </p>
+                                    Virus transduction to hMSC and culture<br/>
-                </li>
+FT cells culture <br/>
-                <li id="2015-05-12" class="" style="opacity: 0.2;">
+                                </p>
-                    <p>
+                            </li>
-                        -Extract the genome of the budding yeast as the template used in the subsequence PCR, yeast stains was Saccharomyces cerevisiae laboratory strain W303 (haploid), and the extraction procedure is based on the fungal genome extraction kit(Omega Bio-Tec).
+                            <li id="2016.08.29-09.04" class="" style="opacity: 0.2;">
-                        <br>
+                                <p>
-                        -Poor yield, partly because the yeast concentration in the suspension is low for genome extraction. The time for yeast incubation should be prolong for extraction next time.
+                                    </br>
-                    </p>
+                                    Virus transduction to 293FT cells<br/>
-                </li>
+                                    Virus transduction to hMSC and culture<br/>
-                <li id="2015-05-14" class="" style="opacity: 0.2;">
+                                </p>
-                    <p>
+                            </li>
-                        -ligation: pBAD-eGFP- B0010;pBAD-EFP- B0010;PBAD-mCherry- B0010.
-                        <br>
-                        -induction and test the intensity of fluorescence
-                        <br>
-                        -HOWEVER our results went bad
-                        <br>
-                        -Extract genome DNA from W303 with yeast genome extraction kit(Omega Bio-Tec).
-                    </p>
-                </li>
-                <li id="2015-05-26" class="" style="opacity: 0.2;">
-                    <p>
-                        -Acquire homologous franking sequence for target integrating site with PCR procedure, using the extracted genome from W303 as a template.
-                        <br>
-                        -Ultraviolet exposure result of the identification gel shows nonspecific amplification. We plan to employ nested PCR.
-                        <br>
-                        -Preservation of W303 strain.
-                        <br>
-                        -Extract genome DNA from W303 with yeast genome extraction kit(Omega Bio-Tec).
-                    </p>
-                </li>
-                <li id="2015-06-01" class="" style="opacity: 0.2;">
+                            <!--04In Vitro Confirmation-->
-                    <p>
+                            <li id="In_Vitro_Confirmation" class="" style="opacity: 0.2;">
-                        -Employ nest PCR to acquire homologous sequence and M phase Promoter sequence using W303 genome.
+                                <p></br>
-                        <br>
+                                    In Vitro Confirmation
-                        -Ultraviolet exposure result of the identification gel shows no target band.
+                                </p>
-                    </p>
+                            </li>
-                </li>
+                            <li id="2016.09.05-11" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+. qPCR: Expression of chemokine receptors and CD markers on the surface of hMSCs<br/>
+                                    <br/>
+.Confirm whether MSCs modified express fluorescent protein.<br/>
+                                    <br/>
+                                    hMSC (CXCR4: eGFP)      hMSC (CXCR5: eGFP)       hMSC (CXCR4: dTomato)<br/>
+                                    <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.12-18" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+.Confirm whether MSCs remain their characteristics after modified.<br/>
+.Transwell: Confirm the function of hMSCs modified by CXCR4/CXCR5<br/>
+                                    <br/>
+. qPCR and western blot: Confirm whether CXCR4/CXCR5 express on the surface of hMSCs<br/>
+                                    <br/>
+                                    <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.19-25" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+.Switch confirmation<br/>
+                                    qPCR: Expression of alpha-SMA on hMSCs before and after adding TGF-β<br/>
+                                    <br/>
+. hMSCs modified by switch plasmid express eGFP after adding TGF-β <br/>
+                                </p>
+                            </li>
-                <li id="2015-06-07" class="" style="opacity: 0.2;">
+                            <!--05Animal Experiment-->
-                    <p>
+                            <li id="Animal_Experiment" class="" style="opacity: 0.2;">
-                        -Employ nest PCR and gradient anneal temperature from 50-60℃ to define the best anneal temperature.
+                                <p></br>
-                        <br>
+                                    Animal Experiment
-                        -Anneal at 60℃ should be better.
+                                </p>
-                    </p>
+                            </li>
-                </li>
+                            <li id="2016.09.05" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    IBD<br/>
+. Carefully shaved a 1cm×1cm filed on the back between the shoulders of the mouse using an electric razor.<br/>
+. While holding the mouse with one hand, smeared the shaved backs with presensitization solution (150μL/20g), which is the mixture of acetone, olive oil and TNBS in 16:4:5 scale.<br/>
+. Control mice are treated with presensitization solution without TNBS.<br/>
+. All the mice were in good condition.<br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.07" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    DTH<br/>
+. Carefully shaved a 1cm×1cm filed on the back between the shoulders of the mouse using an electric razor.<br/>
+. While holding the mouse with one hand, smeared shaved backs with 150μL 0.5% DNFB solution in acetone/olive oil (4:1).<br/>
+. Control mice are treated with identical amount of acetone/olive oil (4:1).<br/>
+. All the mice were in good condition.<br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.11" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    IBD<br/>
+                                    Absolute diet is applied to the mice apart from the water.<br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.12" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Anesthetize the mouse by intraperitoneal injection of 4% chloral hydrate (150μL/20g).<br/>
+                                    Insert the catheter into the colon 4cm proximal to the anus and slowly administer 150μL/20g TNBS/alcohol solution into the colon. Keep the mice with hand down for 60s before returning to the cage. <br/>
+                                    Concerning the damaged colon, we replace normal mice food with jelly to lessen the pain ensuing from sensitization. <br/>
+                                    In the evening, 3 mice are found dead, 2 mice are weak and have less activity, while the others are in good condition.  <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.13" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    IBD<br/>
+                                    Weak mice are dead finally. <br/>
+                                    We weight the mice left. The weight change indicates that 18 of them are modeled successfully. So, we inject 200 μL MSCs in dose of 1*106 into 9 mice from caudal vein. <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.14" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    DTH<br/>
+                                    Challenge right ears with 20μL 0.3% DNFB solution. Two hours after sensitization, inject 200 μL MSCs in dose of 1*106 to 16 mice and PBS of identical amount to another 8 mice of control groups from caudal veins respectively.<br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.15-22" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    IBD<br/>
+                                    All the animals are sacrificed by cervical dislocation. Then we take the colon tissue and measured the length .The colon tissue are kept in PFA or Trizol in EP tube for HE staining or q-PCR. The colon length, DAI score and the concentration of the cytokines will be performed using SPSS. <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.16-21" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    DTH<br/>
+                                    All the animals are sacrificed by cervical dislocation. Then we take the ear tissue and measured the thickness .The ear tissues are kept in PFA or Trizol in EP tube for HE staining or q-PCR. The ear thickness and the concentration of the cytokines will be performed using SPSS.<br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.17" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Start animal experiments for mathematical modeling and cooperation project with Team ShanghaiTechChina_B<br/>
+                                    Day 1, carefully shave a 1cm×1cm filed on the back between the shoulders of the mouse using an electric razor.<br/>
+                                    While holding the mouse with one hand, smear the shaved abdominal skin with presensitization solution (150μL/20g), which is the mixture of acetone, olive oil and TNBS in 16:4:5 scale.<br/>
+                                    Control mice are treated with presensitization solution without TNBS.<br/>
+                                    All the mice are in good condition. <br/>
+                                    <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.17-24" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    Leave the mice until day 7.<br/>
+                                    On day 7, absolute diet is applied to the mice apart from the water.<br/>
+                                    <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.25" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    On day 8, anesthetize the mouse by intraperitoneal injection of 4% chloral hydrate (150μL/20g). Insert the catheter into the colon 4cm proximal to the anus and slowly administer 150μL/20g TNBS/alcohol solution into the colon. Keep the mice with hand down for 60s before returning to the cage. Concerning the damaged colon, we take the jelly for the mice to lessen the pain after modelling. <br/>
+                                </p>
+                            </li>
+                            <li id="2016.09.28-10.05" class="" style="opacity: 0.2;">
+                                <p>
+                                    </br>
+                                    On Day 11, dissolve 5 μg of recombinational EGF in 25 ml saline. Insert 150ug/20g EGF solution into the colon for 6 mice. Sacrifice the narcotic mice with EGF on Day 13.Take the large intestine tissue and measured the length. Keep the large intestine in PFA or Trizol in EP tube for qPCR or HE staining. The colon length, DAI score and the concentration of the cytokines will be performed using SPSS. <br/>
+                                    <br/>
+                                    The mice left were injected 200 μL MSCs in dose of 1*106 into from caudal vein. At 5min, 30 min, 1h, 2h, 4h, 8h, 16h, 24h, 32h, 40h, 48h, 72h after injection, 1 narcotic mouse is taken living image by IVIS spectrum. After that, the mouse are sacrificed and colons are taken and measured for their length. The colons are kept in PFA or Trizol in EP tube for qPCR or HE staining. <br/>
+                                    <br/>
+                                </p>
+                            </li>
-                <li id="2015-06-12" class="" style="opacity: 0.2;">
-                    <p>
-                        -The ligation of pBAD-FR, pBAD-flpe is successful, we tried to insert Cre segment to pSB1A2
-                        <br>
-                        -Employ nest PCR and gradient anneal temperature from 56-66℃ to define the best anneal temperature.
-                        <br>
-                        -The most appropriate anneal temperature should be 65℃.
-                        <br>
-                        -Extract genome DNA from W303 with yeast genome extraction kit(Omega Bio-Tec).
-                    </p>
-                </li>
-                <li id="2015-06-18" class="" style="opacity: 0.2;">
-                    <p>
-                        -Ligation again: pBAD-mCherry-B0010, pBAD-BFP-B0010, pBAD-eGFP-B0015
-                        <br>
-                        -induction and test the intensity of fluorescence
-                        <br>
-                        -Again, we ligated pBAD with FRT
-                    </p>
-                </li>
-                <li id="2015-06-25" class="" style="opacity: 0.2;">
-                    <p>
-                        -We designed qPCR experiments to test whether our system work.
-                    </p>
-                </li>
-                <li id="2015-07-01" class="" style="opacity: 0.2;">
+                            <!--06Submission-->
-                    <p>
+                            <li id="Submission" class="" style="opacity: 0.2;">
-                        -We ordered our IDT gBlocks products.
+                                <p></br>
-                        <br>
+                                    Submission
-                        -We tested whether our ligation products were corrected by sequence and enzyme digestion, and we found that BBa_B0010 in distribution plate was a fake one!
+                                </p>
-                        <br>
+                            </li>
-                        -Acquire M phase promoter sequence with GenStar 2x Taq PCR Star(Mix with Loading Dye) using W303 genome.
+                            <li id="2016.09.26-29" class="" style="opacity: 0.2;">
-                        <br>
+                                <p>
-                        -Clean up the PCR product(M phase promoter nest sequence).
+                                    </br>
-                        <br>
+                                    Using the DNA carrier provided by iGEM, and the DNA to submit, digest and ligate them. But we failed. After analyzing, the reasons we suspect are as followed:<br/>
-                        -Mutation of the plasmid pAUR135.( Muta-direct™ Kit from SBS Genetech Co.,Ltd. SDM-15).
+.The concentration of the DNA is too low to make it,<br/>
-                    </p>
+.The carrier provided by iGEM is useless because the linear carrier has low working efficiency.<br/>
-                </li>
+                                    As a result, we are going to have some meetings to solve it. <br/>
+.Using the part distribution to get the carrier annular carrier by Enzyme digestion, and put the first group of gene into the carrier by ligament.<br/>
+                                    Successful!<br/>
+                                    As a result, we finish the first group of submission, including two genes: CCR7 and CXCR4.<br/>
+                                    <br/>
+                                </p>
+                            </li>
-                <li id="2015-07-10" class="selected" style="opacity: 1;">
+                            <li id="2016.10.04-09" class="" style="opacity: 0.2;">
-                    <p>
+                                <p>
-                        -We found that some of the clones turned red after overnight grown in LB broth. All of these clones were transferred with plasmids contain parts BBa_B0010, indicating that these plasmids were polluted by BBa_J04450.
+                                    </br>
-                        <br>
+.We started working the second group, including CXCR1, CCR5, Luciferease-IRES-eGFP, Luciferase-dTomato-hFTH, CXCR4-IRES-eGFP.<br/>
-                        -Acquire M phase promoter (prefix and suffix added M phase promoter sequence) with PCR procedure, using nest PCR product as template, and gel extraction.
+.After four days’work, we finally finished the second group and put them all into a 96-well format.<br/>
-                        <br>
+                                    <br/>
-                        -Acquire homologous sequence (prefix and suffix added homologous sequence) with PCR procedure, using nest PCR product as template, and gel extraction.
+                                </p>
-                    </p>
+                            </li>
-                </li>
-                <li id="2015-07-15" style="opacity: 0.2;">
+                            <li id="2016.10.09-15" class="" style="opacity: 0.2;">
-                    <p>
+                                <p>
-                        -Add RBS and ssrA(strong) to mCherry by PCR and Extract PCR products by using Gel Extraction Kit.
+                                    </br>
-                        <br>
+.Using the part distribution to get another carrier annular carrier by Enzyme digestion, and put the third group of genes into the carrier by ligament.<br/>
-                        -Double digestion of RBS-mc-ssrA(strong) PCR product and pSB1C3(r) with Spe I and EcoR I. Extract double digestion products by using Gel Extraction Kit.
+                                    The third group includes CXCR5-Luciferease-IRES-eGFP, CXCR4-Luciferease-IRES-eGFP, CXCR4-Luciferase-dTomato-hFTH<br/>
-                        <br>
+.After four days’work, we finally finished the third group and put them all into another 96-well format.<br/>
-                        -Ligation of RBS-mc-ssrA(strong).
+                                    <br/>
-                        <br>
+                                </p>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
+                            </li>
-                        <br>
-                        -We conducted digestion identification and sequencing. No sequencing results were obtained, and the sizes of digestion products were incorrect!
-                        <br>
-                        -Sequencing result of pAUR135 mutant: pAUR135 mutant have  A1041C mutation in CDS of AurR gene, which result in the elimination of Pst I restriction site.
-                        <br>
-                        -Enzyme digestion with FD Pst I (Takara)
-                        <br>
-                        -Extract the product(pAUR135 backbone with pst I sticky end) of 5000+bp
-                        <br>
-                        -End-filling with S1 nuclease.
-                        <br>
-                        -Ligation with T4 DNA polymerase.
-                    </p>
-                </li>
-                <li id="2015-07-18" style="opacity: 0.2;">
-                    <p>
-                        -Pick single colonies of RBS-mc-ssrA(strong), and streak them onto another plate to expand culture.
-                        <br>
-                        -Perform colony PCR of RBS-mc-ssrA(strong).
-                        <br>
-                        -Pick correct colonies of RBS-mc-ssrA(strong), and inoculate a culture of 4 ml LB medium containing chloromycetin.
-                        <br>
-                        -We noted that BBa_B0010(fake) can ligate with our parts, and thus any part ligated with it must be reconstructed!
-                        <br>
-                        -We began to reconstruct our parts, and some of our parts, like Cre and eGFP run out. We amplified BBa_B1006 and BBa_B0015 in DH5a.
-                        <br>
-                        -Transform 5ul T4 ligation product (Jul.15) into 50ul DH5α.
-                        <br>
-                        -Select monoclonal colony and extract plasmid (pAUR135 circular backbone without pst I).
-                        <br>
-                        -Checkout of the removal of Pst I enzyme site.
-                    </p>
-                </li>
-                <li id="2015-07-23" style="opacity: 0.2;">
+                            <div id="grad_left"></div>
-                    <p>
+                            <div id="grad_right"></div>
-                        -Use Plasmid Miniprep Kit to isolate mc-ssrA(strong) from the cultures.
+                            <a href="#" id="next">+</a> <!-- optional -->
-                        <br>
+                            <a href="#" id="prev" style="display: block;">-</a> <!-- optional -->
-                        -We received our booked IDT gBlocks products arrived!
+                         </ul>
-                        <br>
+                    </div>
-                        -We changed our circuits by replacing some devide parts with composite parts. we converted BFP to BBa_E0422(ECFP), converted eGFP to E0840 (GFP with terminator), such changes can accelerate our work
+                </td>
-                        <br>
+            </tr>
-                        -Double enzyme digestion (HF，NEB) with EcoR I and Sac I.
+        </table>
-                        <br>
+        <Br/>
-                        -Gel extraction (Digestion product with EcoR I and Sac I sticky end) With Tiangen gel extraction kit.
-                        <br>
-                        -Anneal of MCS oligonucleotide.
-                         <br>
-                        -Ligation with double digested backbone and MCS anneal product.
-                        <br>
-                        -Transformation with ligation product.
-                        <br>
-                        -no colony found on the plate
-                        <br>
-                        -Double enzyme digestion did not work, as enzyme Sac I has very low(~20%) efficiency when the space between the two enzyme sites is very close(2bp in this experiment).
-                    </p>
-                </li>
-                <li id="2015-07-24" style="opacity: 0.2;">
-                    <p>
-                        -Add RTS sequences (loxp) to the flanks of RBS-mC-ssrA(strong)-1 by PCR.
-                        <br>
-                        -Extract PCR products by using Gel Extraction Kit.
-                        <br>
-                        -Double digestion of loxp-mcss-1 and B0015 (B0015 digest with Xba I and EcoR I, Fragments digest with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.
-                        <br>
-                        -Ligation of loxp-mcss-T.
-                        <br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-                        <br>
-                        -Later sequence result shows the template we used, RBS-mC-ssrA(strong)-1, is not correct. So the clones made by this day are not correct too.
-                        <br>
-                        -We tested and confirmed that mCherry, E0422, E0840, Cre and B1006. They were all corrected.
-                        <br>
-                        -We ligated E0840 with loxP(reverse) and mCherry with B1006. But nearly no clone grown.
-                        <br>
-                        -We obtained some clones with ligation products of mCherry-B1006
-                        <br>
-                        -The first part, BBa_K1641213, was successfully constructed!
-                        <br>
-                        -Parts construction
-                        <br>
-                        -Adjust double enzyme digestion into two single digestions, first Sac I then EcoR I.
-                        <br>
-                        -MSC modification fails.
-                        <br>
-                        -Change MSC modification primer using Kpn I and EcoR I as sticky end.
-                        <br>
-                        -Double enzyme digestion with Kpn I and EcoR I and then ligate with MCS annealed product.
-                        <br>
-                        -Transformation.
-                        <br>
-                        -Extract plasmid: pAUR135-KE.
-                        <br>
-                        -Sequencing result shows incorrect ligation.
-                    </p>
-                </li>
-                <li id="2015-07-29" style="opacity: 0.2;">
-                    <p>
-                        -Add RTS sequences (loxp) to the flanks of RBS-mC-ssrA(strong)-2 by PCR. Extract PCR products by using Gel Extraction Kit.
-                        <br>
-                        -Ligated E0840 with loxP and pBAd-flpe-ECFP. We tried several methods of ligation. Preliminary "2A" assembly mthod was developed. However, colony PCR showed high false positive rate of these clones.
-                    </p>
-                </li>
-                <li id="2015-08-01" style="opacity: 0.2;">
-                    <p>
-                        -Double digestion of loxp-mcss and B0015. (B0015/B1006 digested with Xba I and EcoR I, Fragments digested with Spe I and EcoR I).Extract double digestion products by using Gel Extraction Kit.
-                        <br>
-                        -Ligation of loxp-mcss-T.
-                        <br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-                        <br>
-                        -Pick single colonies of loxp-mcss-T, and streak them onto another plate to expand culture.
-                    </p>
-                </li>
-                <li id="2015-08-04" style="opacity: 0.2;">
-                    <p>
-                        -Perform colony PCR of loxp-mcss-T and record the correct colonies.
-                        <br>
-                        -Pick correct colonies of loxp-mcss-T, and inoculate a culture of 4 ml LB medium containing chloromycetin.
-                        <br>
-                        -Use Plasmid Miniprep Kit to isolate loxp-mcss-T from the cultures.
-                        <br>
-                        -We used PCR to reconstruct RBS-Cre-ssrA, and inserted this fragment into pSB backbone.
-                        <br>
-                        -All clones grown well! But somebody placed our enzymes out of refrigerator overnight. We went crazy! So our process was delayed.
-                        <br>
-                        -We placed the gBlocks products into pSB backbones. Most of them were successful. Also we gained some correct clones of loxP-GFP(BBa_K16416209).
-                        <br>
-                        -Ligations: Cre with mCherry and K1641206 with flpe-ECFP
-                        <br>
-                        -pAUR135-MCS modification
-                        <br>
-                        -Double enzyme digestion with Kpn I and EcoR I and then ligate with MCS annealed product.
-                        <br>
-                        -Transformation;Extract plasmid: pAUR135-MCS-KE
-                        <br>
-                        -Clone biobrick:KRE9UTR;GFP-PEST191;SV40NLS;Venus YFP
-                        <br>
-                        -Patch:bxb1gp35 RFC25optimized-puc19
-                        <br>
-                        -Transformation and Plasmid Preparation-MCS-KE
-                        <br>
-                        -Sequencing confirmed.
-                    </p>
-                </li>
-                <li id="2015-08-10" style="opacity: 0.2;">
-                    <p>
-                        -Again, we tried to ligate the short intermediates with the long parts:pBAD(reverse) with FPs, etc.
-                        <br>
-                        -We successfully ligated pBAD(reverse) with flpe-ECFP.
-                        <br>
-                        -We used endonuclease and sequencing skills to test the correctness of ligation
-                        <br>
-                        -Clone cyclic promoters;PCR :YMR013C/Cln3p/Met16p from yeast genome add attB/P sites, prefix and suffix
-                        <br>
-                        -transformation and plasmid preparation:MCS-KE
-                        <br>
-                        -Assemble:Pgal+kozak-1C3;bxb1gp35+SV40NLS-1C3;;Pbad-RBS-1C3
-                        <br>
-                        -Double digestion, ligation and transformation
-                        <br>
-                        -Mutation:MCS-KE to RFC25 optimized
-                        <br>
-                        -Substrate digested via Fast Digest-Dpn I.
-                        <br>
-                        -product transformed in TOP10 50, Amp
-                    </p>
-                </li>
-                <li id="2015-08-17" style="opacity: 0.2;">
-                    <p>
-                        -Double digestion of loxp-mc-1, loxp-mc-2, B0015 and CG, CGS, B1006 (B0015/B1006 digested with Xba I and EcoR I. Fragments digested with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.
-                        <br>
-                        -Ligation of loxp-mc-1-T, loxp-mc-2-T and CGT, CGST.
-                        <br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.
-                        <br>
-                        -Use Plasmid Miniprep Kit to isolate I0500 from the cultures.
-                        <br>
-                        -We ligated pBAD(forward) with other parts contains FPs
-                        <br>
-                        -We test whether pBAD and FPs can worked, but we met high false positive rates.
-                        <br>
-                        -We found that the ECFP sequence of our BBa_K1641209 sample was reverse! Oh my God!
-                        <br>
-                        -Clone biobrick:ECFP;EYFP;mRFP1
-                        <br>
-                        -PCR:reversed mRFP1;reversed KRE9UTR
-                        <br>
-                        -Plasmid preparation: pAUR135 RFC25 optimized
-                        <br>
-                        -Assemble:reversed KRE9UTR+reversed mRFP1;eCFP+KRE9UTR
-                        <br>
-                        -Double digestion, ligation and transformation
-                        <br>
-                        -Add sequence via PCR:
-                        prefix-RBS-eCFP-terminatior-frt;loxP-RBS-Cre-ssrA;prefix-RBS-GFP-Terminator-loxp;prefix-ECFP-frt-suffix;prefix-loxP-Cre-suffix;prefix-GFP-loxp-suffix
-                        <br>
-                        -Assemble:Met16p-eCFP;YMR013C-eCFP;Loxp-cre+gfp-loxp;frt-flpe-cfp-frt
-                        <br>
-                        -Transformation.
-                        <br>
-                        -Plasmid preparation.
-                    </p>
-                </li>
-                <li id="2015-08-25" style="opacity: 0.2;">
-                    <p>
-                        -Double digestion of dre, vika, scre, vcre, pSB1C3(r) with Spe I and EcoR I. Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of dre, vika, scre, vcre and pSB1C3(r).<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -Pick single colonies of dre(1c3), vika(1c3), vcre(1c3), scre(1c3), and streak them onto another plate to expand culture. <br>
-                        -Perform colony PCR of dre(1c3), vika(1c3), vcre(1c3), scre(1c3) and record the correct colonies.<br>
-                        -Pick correct colonies of dre(1c3), vika(1c3), vcre(1c3), scre(1c3), and inoculate a culture of 3 ml LB medium containing chloromycetin<br>
-                        -Add RTS sequences(loxp) to the flanks of RBS-mC-ssrA(strong) by PCR. Electrophoresis result didn’t shows correct bands.<br>
-                        -Use Plasmid Miniprep Kit to isolate dre(1c3), vika(1c3), vcre(1c3), scre(1c3) from the cultures.<br>
-                        -We tried to amplify BBa_K1641203, BBa_K1641201, BBa_K1641209 <br>
-                        -We ligated mCherry with BBa_K1641208.<br>
-                        -Assemble:reversed KRE9UTR+reversed mRFP1;eCFP+KRE9UTR<br>
-                        -Restriction analysis:loxp-cre-gfp-loxp<br>
-                        -Assemble:YFP-KRE9UTR;reversed KRE9UTR+reversed mRFP1<br>
-                        -Sequencing:loxp-cre-gfp-loxp
-                    </p>
-                </li>
-                <li id="2015-08-28" style="opacity: 0.2;">
-                    <p>
-                        -Prepare RBS-Flpe-fc by PCR.<br>
-                        -Use Plasmid Miniprep Kit to isolate CG, CGS, B0015, B1006, I0500 from the cultures.<br>
-                        -Add RTS sequences(loxp) to the flanks of RBS-mC-ssrA(strong) by PCR. Electrophoresis result didn’t shows correct bands.<br>
-                        -Add RTS sequences(FRT, Rox, SloxpM1, Vloxp, vox) to the flanks of RBS-mC-ssrA(strong) by PCR. Electrophoresis result didn’t shows correct bands.<br>
-                        -We had many colony PCR experiments to find a correct clone, we did.<br>
-                        -We eventually had a real BBa_K1641209. Also we have correct BBa_1641203 and BBa_K1641201. So we can use these parts to construct the Circuit2, which was improved on the basis of Circuit1. <br>
-                        -clone and assemble:loxp-cre-gfp-loxp;frt-flpe-cfp-frt<br>
-                        -sequencing inconsistant
-                    </p>
-                </li>
-                <li id="2015-09-01" style="opacity: 0.2;">
-                    <p>
-                        -Double digestion of PCG-1, PCGS-1, B1006. (B1006 digested with Xba I and EcoR I, fragment digested with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of PCGT, PCGST.<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -Perform 20ul system PCR to test if the primers of FRT, Rox, SLM, VL, vox work.<br>
-                        -Add RTS sequences to the flanks of FRT, Rox, SLM, VL, vox by PCR. Extract PCR products by using Gel Extraction Kit.<br>
-                        -Double digestion of FRT, Rox, SLM, VL, vox and pSB1C3(r) with EcoR I and Pst I. Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of FRT, Rox, SLM, VL, vox.<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -Use Plasmid Miniprep Kit to isolate PCGT-1, PCGST-1, pSB3k3 from the cultures.<br>
-                        -Double digestion of PCGT-1, PCGST-1 and pSB3k3. Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of PCGT-1(3k3), PCGST-1(3k3).<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+kan.<br>
-                        -Perform colony PCR of FRT-mcsm, Rox-mcsm, VL-mcsm, vox-mcsm.<br>
-                        -Pick correct colonies of FRT-mcsm, Rox-mcsm, VL-mcsm, vox-mcsm, and inoculate a culture of 4 ml LB medium containing chloromycetin.<br>
-                        -Perform colony PCR of RBS-mc-ssrA(strong).<br>
-                        -Pick correct colonies of RBS-mc-ssrA(strong), and inoculate a culture of 4 ml LB medium containing chloromycetin.<br>
-                        -We successfully ligated pBAD(reverse) with flpe-ECFP and we also got correct BBa_K1641214.<br>
-                        -We tried to induce BBa_K1641214 in DH5a and Top10 strains with L-arabinose in LB broth, then test the intensity of fluorescence
-                    </p>
-                </li>
-                <li id="2015-09-06" style="opacity: 0.2;">
-                    <p>
-                        -Use Plasmid Miniprep Kit to isolate FRT-mcsm-3, Rox-mcsm-3, SLM-mcsm-4, VL-mcsm-4, vox-mcsm-2,VGC-3, PCGT(3k3), PCGST(3k3), mc-ss-2 from the cultures.<br>
-                        -Double digestion of ScGS-1, VGS-3, VcGS-4, FGS-1, DGS-1 and I0500. Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of PDGS, PFGS, PVcGS, PVGS, PScGS.<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -Perform colony PCR of PDGS, PFGS, PScGS, PVcGS.<br>
-                        -Pick correct colonies of PDGS, PFGS, PScGS, PVcGS, and inoculate a culture of 4 ml LB medium containing chloromycetin.<br>
-                        -Double digestion of RT-mcsm-3, Rox-mcsm-3, SLM-mcsm-4, VL-mcsm-4, vox-mcsm-2 and B0015. (B0015 digested with Xba I and EcoR I, fragment digested with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of FRT-mcsm-T, Rox-mcsm-T, SLM-mcsm, VL-mcsm-T.<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -Use Plasmid Miniprep Kit to isolate PDGS-2, PFGS-5, PVcGS-1, PVGS-4, p23101, B0015, loxp-mcsm-Inv-rep from the cultures.<br>
-                        -Double digestion of PDGS-2, PFGS-5, PVcGS-1, PVGS-4 and B1006. (B1006 digested with Xba I and EcoR I, fragment digest with Spe I and EcoR I). Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of PDGST, PFGST, PScGST, PVcGST.<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -Transform PCGT with loxp-mcsm-Inv-rep, PCGST with loxp-mcsm-Inv-rep, plate the transformed cells on LB+kan+cm.
-                    </p>
-                </li>
-                <li id="2015-09-10" style="opacity: 0.2;">
-                    <p>
-                        -Use Plasmid Miniprep Kit to isolate FRT-mcsm-T, Rox-mcsm-T, SLM-mcsm, VL-mcsm-T from the cultures.<br>
-                        -Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of P-FRT-mcsm-T(FRT-reporter), P-Rox-mcsm-T(Rox-reporter), P-SLM-mcsm(SLM-reporter), P-VL-mcsm-T(VL-reporter).<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -Use Plasmid Miniprep Kit to isolate PDGST-5, PFGST-1, PScGST-4, PVcGST-1, VGS-4, VGS-5 from the cultures.<br>
-                        -Double digestion of PDGST-5, PFGST-1, PScGST-4, PVcGST-1 and pSB3K3. Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3). <br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+kan.<br>
-                        -pcr (add sequence):prefix-RBS-flpe-FC, FC-Mcherry-T7-frt-suffix from biobrick<br>
-                        -overlap pcr:RBS-flpe-FC-Mcherry
-                    </p>
-                </li>
-                <li id="2015-09-12" style="opacity: 0.2;">
-                    <p>
-                        -Pick single colonies of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3), and streak them onto another plate to expand culture.<br>
-                        -Perform colony PCR of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3). Colony No. 2, 3, 4, 6 of PDGST-5(3k3), No. 2, 3, 4, 5 of PFGST-1(3k3), No. 1, 2, 3, 4, 5, 6 of PScGST-4(3k3), No. 3, 4, 5, 6 of PVcGST-1(3k3) are correct.<br>
-                        -Pick correct colonies of PDGST-5(3k3), PFGST-1(3k3), PScGST-4(3k3), PVcGST-1(3k3), and inoculate a culture of 4 ml LB medium containing kanamycin. <br>
-                        -Use Plasmid Miniprep Kit to isolate PDGST-5-2, PFGST-1-2, PScGST-4-1, PVcGST-1-4 from the cultures.<br>
-                        -Double digestion of PDGST-5-2, PFGST-1-2, PScGST-4-1, PVcGST-1-4 and pSB1K3 with EcoR I and Pst I. Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of PDGST-5-2(1k3), PFGST-1-2(1k3), PScGST-4-1(1k3), PVcGST-1-4(1k3).<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+kan.<br>
-                        -Transform PDGST-2(3k3) with Rox-repoter, PFGST-4(3k3) with FRT-reporter, PScGST-1(3k3) with SLM-repoter, PVcGST-2(3k3) with vox-repoter, plate the transformed cells on LB+kan+cm. <br>
-                        -We detected ECFP in M9 broth!<br>
-                        -BBa_K1641225 correctly constructed!<br>
-                        -pcr (add sequence):prefix-flpe-FC, FC-Mcherry-T7TE-frt-suffix from biobrick<br>
-                        -overlap pcr:flpe-FC-Mcherry;overlap pcr worked<br>
-                        -assemble:Pbad-frt-RBS-flpe-FC-Mcherry-T7TE-frt in pSB1A2
-                    </p>
-                </li>
-                <li id="2015-09-14" style="opacity: 0.2;">
-                    <p>
-                        -Use Plasmid Miniprep Kit to isolate cgs, vgs, gp38 from the cultures.<br>
-                        -Pick single colonies of PDGST-4(1k3), PFGST-4(1k3), PScGST-4(1k3), PVcGST-1(1k3) , and streak them onto another plate to expand culture.<br>
-                        -Perform colony PCR of PDGST-4(1k3), PFGST-4(1k3), PScGST-4(1k3), PVcGST-1(1k3). Colony No. 1, 2, 3, 4, 5, 6 of PDGST-4(1k3), No. 2, 4, 6, 7 of PFGST-4(1k3), No. 1, 2, 3, 4, 5, 6 of PScGST-4(1k3), No. 1, 2, 3, 4, 5, 6 of PVcGST-1(1k3) are correct.<br>
-                        -Pick correct colonies of PDGST-4(1k3), PFGST-4(1k3), PScGST-4(1k3), PVcGST-1(1k3), and inoculate a culture of 4 ml LB medium containing kanamycin.<br>
-                        -Use Plasmid Miniprep Kit to isolate PDGST(1k3), PFGST(1k3), PScGST(1k3), PVcGST(1k3), Test-mc-T, Test-rep, p23101, I0500, B1006 from the cultures.<br>
-                        -Double digestion of FRT-mcsm-T, Rox-mcsm-T, SLM-mcsm-T, VL-mcsm-T (Spe I and Pst I) and p23101(Xba I and Pst I). Extract double digestion products by using Gel Extraction Kit.<br>
-                        -Ligation of p-FRT-mcsm-T, p-Rox-mcsm-T, p-SLM-mcsm-T and p-VL-mcsm-T.<br>
-                        -Transform the ligation reactions into standard bacterial cloning cells(DH5α), plate the transformed cells on LB+cm.<br>
-                        -We conducted fluorescence measurements to confirmed whether BBa_K1641225 can work. We still tried to construct a full plasmid in Circuit 2.<br>
-                        -pcr (add sequence):prefix-loxP-RBS-Cre-FC, FC-eGFP-T7TE-loxP-suffix from biobrick<br>
-                        -overlap pcr:loxP-RBS-Cre-FC-eGFP-T7TE-loxP;overlap pcr worked<br>
-                        -assemble:Pbad-frt-RBS-flpe-FC-Mcherry-T7TE-frt-loxP-RBS-Cre-FC-eGFP-T7TE-loxP in pSB1A2
-                    </p>
-                </li>
-                <div id="grad_left"></div>
-                <div id="grad_right"></div>
-                <a href="#" id="next">+</a> <!-- optional -->
-                <a href="#" id="prev" style="display: block;">-</a> <!-- optional -->
-            </ul>
-        </div>
      </div>
 </div>
@@ Line 1,170: / Line 1,346: @@
                  var whichIssue = $(this).text();
                  var currentIndex = $(this).parent().prevAll().length;
+                console.log('in event');
                  // moving the elements
                  if(settings.orientation == 'horizontal') {
@@ Line 1,178: / Line 1,355: @@
                  $(settings.issuesDiv+' li').animate({'opacity':settings.issuesTransparency},{queue:false, duration:settings.issuesSpeed}).removeClass(settings.issuesSelectedClass).eq(currentIndex).addClass(settings.issuesSelectedClass).fadeTo(settings.issuesTransparencySpeed,1);
                  // prev/next buttons now disappears on first/last issue | bugfix from 0.9.51: lower than 1 issue hide the arrows | bugfixed: arrows not showing when jumping from first to last date
-                 if(howManyDates == 1) {
+                 if (howManyDates == 1) {
                      $(settings.prevButton+','+settings.nextButton).fadeOut('fast');
-                 } else if(howManyDates == 2) {
+                 } else if (howManyDates == 2) {
                      if($(settings.issuesDiv+' li:first-child').hasClass(settings.issuesSelectedClass)) {
                          $(settings.prevButton).fadeOut('fast');
                          $(settings.nextButton).fadeIn('fast');
                      }
-                     else if($(settings.issuesDiv+' li:last-child').hasClass(settings.issuesSelectedClass)) {
+                     else if ($(settings.issuesDiv+' li:last-child').hasClass(settings.issuesSelectedClass)) {
                          $(settings.nextButton).fadeOut('fast');
                          $(settings.prevButton).fadeIn('fast');
@@ Line 1,435: / Line 1,612: @@
          });
+        $(".tline-trigger").click(function(event) {
+            event.preventDefault();
+            var link = $(this).attr("href"), obj,
+                    s= "#dates a[href='" + link + "']";
+            console.log(link);
+            console.log(s);
+//            switch (link) {
+//                case '#Story_Began' : obj = $("#date a[href='#Story_Began']");
+//            }
+            console.log($(s));
+            $(s).click();
+        });
 //        var prev = $('#prev'), next = $('#next');

Difference between revisions of "Team:SYSU-MEDICINE/Notebook"

Revision as of 15:38, 17 October 2016

Notebook