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 Began.

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

	New primers without attB * template cDNA 100bp marker positive control CCR7 1kb marker
	New primers without attB * new template cDNA 100bp marker CXCR5 1kb marker

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

100bp marker Positive control CCR7 CCR7 No CXCR5s

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

CCR7 CXCR5 positive control 100bp marker

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.

Positive control CXCR5 * 2 CCR7 * 1 1kb marker

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

100bp marker CXCR5 CCR7

11. PCR: TA primers（CXCR1, CXCR3, CXCR4, CCR2, CCR5）* template，Agarose Gel Electrophoresis 10μl
Surprise: CCR2, CXCR3

100bp marker CXCR3 CCR2 Positive control 1kb marker

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

Positive control CXCR5 * 1 CCR7 * 2 1 kb marker

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

CXCR3 CCR2 Positive control 1kb marker

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

Positive control 1kb marker No results.

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

1kb marker CXCR3 100bp marker

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

Positive control CXCR5 * 2 1kb marker

100bp marker CCR7 * 2 Positive control

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

CXCR4 100bp marker

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

100bp marker CXCR4 positive control 1kb marker

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

Positive control CXCR1 CXCR3 CCR2 CCR5 1kb marker

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

Positive control CXCR1 CXCR3 CCR2 CCR5 1kb marker

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

1kb marker Positive control CXCR4 * 2

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.

Positive control CXCR1 CCR5 1kb marker

13. Extract the plasmids (TA-CXCR1/ CXCR4/ CCR5) from them.
14. PCR：attB primers (CXCR1, CXCR4, CCR5) * plasmid templates, and Agarose Gel Electrophoresis 10μl

CXCR1, CXCR4, CCR5, Positive control 1kb marker

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

CXCR1, CXCR4, CCR5, 1kb marker

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.)

Positive control CXCR1 * 1 CXCR4 * 2 CCR5 * 2 1kb marker 100bp marker

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.

CCR2 Positive control 1kb marker

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

1kb marker CCR2 others Positive control 100bp marker

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.)

100bp marker CCR2-1064 * 2

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

100bp marker CXCR3 Positive control 1kb marker

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

CXCR3 100bp marker

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

100bp marker (tract 2) CXCR1 (tract4,5)，CXCR5 (tract7,8), CCR2 (tract 10, 11) CCR5(tract 12,13)，

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

100bp marker (tract 5) CXCR5 (tract2), CCR5(tract 3)，

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

100bp marker (tract 1) CXCR1-86bp (tract2), CXCR1-1000+bp (tract 3)

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

100bp marker (tract 1) CXCR1-overlap (tract2), CXCR1-postive control (tract 3)

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

100bp marker (tract 1) CXCR1-postive control (tract2), CXCR1-adding 32bp (tract 3)

6 PCR: fragment IRES to dTomato
Agarose Gel Electrophoresis

100bp marker (tract 1) IRES (tract2), dTomato (tract 3)

RESULTS: NO

1 PCR: the second time (adding 34bp to the posterior+32bp fragment)
Agarose Gel Electrophoresis

100bp marker (tract 1) CXCR1-34bp+posterior+32bp (tract2), CXCR1-positve control (tract 4)

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.

100bp marker (tract 1) CXCR1 (tract2-6), CXCR1-positve control (tract 7)

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

100bp marker (tract 1) CXCR4-LR (tract2-6), CXCR4-positve control (tract 7)

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

100bp (tract1) CXCR5 point mutation (tract 2) CXCR5 positive control (tract 3)

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

100bp marker (tract 1) CXCR5-point mutation-BP (tract2-4), CXCR5-positve control (tract 5)

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

α-SMA promoter (tract1-2), 100bp marker (tract 3)

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

Pup α-SMA promoter (tract1-3), 1kbp marker (tract 4)

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

100bp marker (tract 1) a-SMA promoter-eGFP (tract2-7),

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

100bp marker (tract 1) CXCR5-R1 (tract10), F1-luciferase (tract11)

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.

F1-luciferase-T3 (tract1-4) CXCR5-R1-T3 (tract5-8), 100bp marker (tract 9)

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

CXCR5-R2 (tract1), F2-luciferase (tract2) 100bp marker (tract 3)

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

CXCR5-Luciferase attB (tract1), 100bp marker (tract 2)

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

Positive control (tract1), CXCR5-luciferase BP (tract 2-4) 100bp marker (tract 5)

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

100bp marker (tract 1) CXCR5-luciferase-ires-eGFP-LR (tract2-5), positve control (tract 6)

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

CXCR4-R1 (tract1), 100bp marker (tract 3)

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.

CXCR4-R1-T3 (tract1-4), 100bp marker (tract 5)

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

CXCR4-R2 (tract1), 1kbp marker (tract 2)

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

CXCR4-Luciferase attB (tract1), 100bp marker (tract 4)

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

100bp marker (tract 1) Positive control (tract3), CXCR5-luciferase BP (tract 4)

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

100bp marker (tract 1) CXCR5-luciferase-ires-eGFP-LR (tract2-5), positive control (tract 6)

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

	1kbp marker (tract 1) luciferase+dtomato (anterior fragment) (tract2-3), 100bp marker(track6)
	100bp marker (tract 1) dtomato(posterior fragment)+hFTH (tract3),

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

Luciferase-dtomato(point mutation)-hFTH (tract1), 100bp marker (tract 2)

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

100bp marker (tract 1) dtomato(posterior fragment)+hFTH(anterior fragment) (tract2), hFTH(posterior fragment)(tract3)

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

dtomato+hFTH(point mutation) (tract1), 100bp marker (tract 3)

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

100bp marker(tract1) Luciferase-dtomato(point mutation)-hFTH(point mutation) (tract1-2)

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

IRES (tract1-2) 100bp marker(tract3)

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

IRES-luciferase-dtomato(point mutation)-hFTH(point mutation) (tract1-2) 100bp marker(tract3)

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

100bp marker (tract 1) IRES-luciferase-dtomato-hFTH-vector ptail (tract2-5), Positive control (tract 5)

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

100bp marker (tract 1) CXCR5-luciferase-ires-eGFP-LR (tract2-5), positve control (tract 6)

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.

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.