Team:Linkoping Sweden/Experiments
Contents
Experiments
Overview on Laboration
Week 1
14 June
- First day at the lab! Making Hutner’s trace elements.
Week 2
21 June
- Making SOC medium, LB medium, LB agar and chloramphenicol plates.
Week 3
27 June
- Transformation of [http://parts.igem.org/Part:BBa_E1010 BBa_E1010] to test transformation protocol. Observation: The transformation was successful.
28 June
- Control of competent cells.
29 June
- Transformation of BBa_E1010 to super competent XL-1. Observation: The transformation was successful.
30 June
- Making new E.Coli Calcium chloride competent cells.
1 July
- Making solutions for TAP and TRIS medium.
- Cultivation of successful transformants.
Week 4
4 July
- Making LB medium and LB agar.
- Plasmid preparation of succeded transformants week 3.
- Test cultivation of algae.
5 July
- Making agar plates.
- Starting standard assembly of construct; digestion and ligation of pLIP ([http://parts.igem.org/Part:BBa_K2095000 BBa_K2095000]), U6 promoter, RBCS2 terminator ([http://parts.igem.org/Part:BBa_K2095002 K2095002]) and LIP-RFP ([http://parts.igem.org/Part:BBa_K2095003 BBa_K2095003]).
- Competent cell test.
- First algae cultivation.
6 July
- Transformation on U6, pLIP, LIP-RFP and RBCS2 terminator. Observation: Colonies for U6, pLIP and LIP-RFP were detected.
7 July
- Cultivation of U6, pLIP, LIP-RFP colonies in LB medium with chloramphenicol. Observation: Transformation of insert did not succeed
Week 5
11 July
- PCR on Cas9.
12 July
- Gel electrophoresis on Cas9 to see if the PCR was successful. Observation: No bands were obtained for Cas9.
14 July
- PCR on pSB1C3 for more product.
15 July
- Gel electrophoresis on pSB1C3. Observation: No bands were obtained on the gel.
Week 6
18 July
- PCR and gel electrophoresis on pSB1C3. Observation: No bands were obtained on the gel.
20 July
- PCR and gel electrophoresis on pSB1C3. Observation: Correct bands were obtained - PCR on pSB1C3 succeeded.
22 July
- Digestion, ligation and transformation on pLIP, U6, RBCS2 terminator, Cas9, LIP-RFP, sgRNA and pSB1C3.
Week 7
25 July
- PCR and recultivation of transformed pLIP and RBCS2 terminator colonies.
- PCR purification of pSB1C3.
26 July
- Gel electrophoresis on pSB1C3, pLIP, RBCS2. Observation: No bands were obtained on the gel.
- Digestion and ligation on LIP-RFP and pSB1C3.
27 July
- New project approach - Left standard assembly for Gibson assembly
- Transformation of LIP-RFP and pSB1C3.
Week 8
1 August
- Cultivation of hygromycin-containing bacteria in order to isolate hygromycin which would be utilized to test electroporation in C.Reinhardtii
- Gel electrophoresis on pLIP and RBCS2 terminator. Observation: Bands were obtained on the gel at 700 bp, indicates insert.
3 August
- Plasmid preparation of pLIP, RBCS2 terminator and hygromycin. Observation: Turned out to be incorrect later on.
- Transformation of LIP-RFP, U6, sgRNA and Cas9. Observation: 4 colonies for sgRNA and 4 colonies for LIP-RFP were obtained, but unfortunately no colonies for Cas9 and U6.
4 August
- Making TAP medium for cultivation of algae in the dark.
Week 9
8 August
- PCR and recultivation on transformed LIP-RFP and sgRNA colonies.
- First algae cultivation in darkness.
9 August
- Transformation of RBCS2 terminator and Cas9.
- Gel electrophoresis on LIP-RFP and sgRNA. Observation: No bands were obtained.
10 August
- PCR on LIP-RFP and sgRNA.
- Gel electrophoresis on LIP-RFP. Observation: Bands slightly above 1000 bp were obtained. LIP-RFP with primers should be at 1300 bp. It is looking good.
11 August
- PCR on U6 and Cas9.
- Gel electrophoresis on sgRNA. Observation: No correct bands were obtained.
12 August
- Gel electrophoresis on Cas9 and U6. Observation: No correct bands were obtained.
Week 10
15 August
- Preparation of TAP agar. Observation: Because of difficulties with the gas no plates could be performed today.
- Gel electrophoresis on RBCS2 terminator, pLIP, hygromycin and pSB1C3. Observation: No correct bands were obtained except for pSB1C3.
- New cultivation of hygromycin-containing bacteria.
16 August
- Cultivation of sgRNA, LIP-RFP and U6 transformants.
- PCR on Cas9 and hygromycin.
- Gel electrophoresis on Cas9 and hygromycin. Observation: The gel showed a weak band for Cas9 around 4000 bp.
17 August
- Plasmid preparation of LIP-RFP, U6 and sgRNA. Observation: Turned out to be incorrect later on.
18 August
- Cultivation of algae for transformation. Observation: It took 5 days for the wild type algae to reach OD = 1,757. The mutant algae evaporated.
- Making TAP agar plates.
- Making TAP Hyg. plates.
Week 11
22 August
- Cultivation of LIP-RFP and hygromycin.
23 August
- Plasmid preparation of LIP-RFP and hygromycin-containing bacteria.
- PCR on Cas9 and pSB1C3.
- New cultivation of algae in the dark.
24 August
- PCR on LIP-RFP, hygromycin and pSB1C3.
- Gel electrophoresis on Cas9, hygromycin, LIP-RFP and pSB1C3. Observation: Bands for Cas9 and hygromycin were obtained.
- PCR purification of Cas9.
25 August
- Gel electrophoresis on pSB1C3. Observation: Bands were detected at 2000 bp which corresponds to the size of pSB1C3.
- PCR purification of pSB1C3.
- First Gibson Assembly!
- Transformation of Gibson Assembly. Observation: Colonies were obtained!
- PCR on Cas9, hygromycin, pSB1C3.
26 August
- PCR and recultivation of Gibson Assembly transformants.
- Chloramphenicol plates.
- Gel electrophoresis on PCR product from Gibson Assembly, Cas9, hygromycin and pSB1C3. Observation: Bands were detected for all the DNAs!
Week 12
29 August
- Gel electrophoresis on Gibson Assembly colonies. Observation: Band at 5500 bp was obtained, 7000 bp would indicate complete insert.
- A new digestion on LIP-RFP.
- PCR screening on Gibson Assembly colonies.
30 August
- PCR screening on Gibson Assembly colonies.
- Cultivation of Gibson Assembly colonies on new plates.
- Ligation and transformation on LIP-RFP with pSB1C3.
- New Gibson Assembly transformation.
31 August
- Gel electrophoresis on Gibson Assembly colonies. Observation: The bands indicate that there is no insert.
- Plasmid preparation of Gibson Assembly colony (2016-08-29).
1 September
- PCR on plasmid prepared U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin.
- Gel electrophoresis on U6, RBCS2 terminator, pLIP, LIP-RFP, sgRNA and hygromycin Observation: No bands were obtained.
2 September
- Second Gibson Assembly.
- Gibson transformation.
- Transformation of LIP-RFP.
3 September
- PCR and gel electrophoresis on Gibson colonies. Observation: No bands were obtained on the gel.
- Digestion and ligation of pLIP, U6, RBCS2 terminator, sgRNA and pSB1C3.
4 September
- PCR and gel electrophoresis on Gibson colonies. Observation: Band from one colony (colony 8) indicates correct insert!
Week 13
5 September
- PCR on old colonies of LIP-RFP.
- Making LB medium.
- Gel electrophoresis on LIP-RFP and plasmid prepared Gibson colonies.
6 September
- Cultivation of colony 8 (Gibson Assembly) with hygromycin in the LB media.
- Screening of colonies from Gibson Assembly. Observation: Bands were obtained, but no band was at 7000 bp.
7 September
- PCR and gel electrophoresis on hygromycin.
8 September
- Plasmid preparation of Gibson Assembly colony 8.
- Screening on Gibson colonies.
9 September
The sequence from colony 8 was obtained. We did not insert hygromycin but instead YFP was inserted, this due to a 50% chance of amplifying either hygromycin or YFP when using PCR. Cas9 and pLIP were inserted successfully!
10 September
- Cultivation of algae mutants and Gibson colony 8.
- Gel electrophoresis on Gibson colonies and plasmid prepared colony 8. Observation: The plasmid preparation of Gibson colony 8 showed good bands.
11 September
- PCR on Gibson colonies.
- Recultivation of Gibson Assembly colonies on new plates.
Week 14
13 September
- OD measurements on the algae.
- Gel electrophoresis on the PCR product from 11/9 - 16. Observation: No correct bands were obtained.
14 September
- Dilution of the algae.
- Making TAP-40mM sucrose.
- Plasmid preparation of Gibson Assembly colony 8.
15 September
- Digestion of Gibson colony 8, for the electroporation.
16 September
- Electroporation on algae with Gibson colony 8 as the vector Observation: The algae have grown well.
17 September
- PCR screening on Gibson colonies.
- Continuation on the electroporation from previous day.
18 September
- Gel electrophoresis on Gibson colonies from previous day.
Week 15
19 September
- The sequence from Gibson colony 8 was obtained. Observation: Looks like we did not insert U6 and sgRNA.
20 September
- Third Gibson Assembly.
- Gibson transformation.
21 September
- PCR screening on the third Gibson Assembly.
- Gel electrophoresis on the PCR product from today. Observation: Band were obtained at 300 bp and 2000 bp.
22 September
- Gel electrophoresis on PCR product from previous day. Observation: No correct bands were obtained.
- A new Gibson Assembly but only on pSB1C3 with pLIP, LIP-RFP, U6 and RBCS2 terminator respectively.
- Gibson transformation.
23 September
- Gel electrophoresis on the obtained colonies from the third Gibson. Observation: No bands were detected on the gel.
- PCR of Gibson with pLIP, LIP-RFP, U6 and RBCS2 terminator.
- Screening of YFP transformed algae. Observation: No proof that the transformation worked.
24 September
- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. Observation: Bands were obtained at 500 bp for RBCS2 terminator, 600 bp for pLIP and 1500 bp for LIP-RFP. No result for U6.
- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: The bands indicate that there was no insert.
- PCR on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP.
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
25 September
- PCR on the colonies obtained from the third Gibson.
- Gel electrophoresis on Gibson with U6, RBCS2 terminator, pLIP and LIP-RFP. Observation: Bands were obtained for all the DNA fragments except from U6
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
Week 16
26 September
- PCR on U6 colonies.
- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: The bands indicate that there is no insert.
- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.
27 September
- Plasmid preparation nr 2 of pLIP, LIP-RFP and RBCS2 terminator.
- Gel electrophoresis on U6 colonies. Observation: No results.
- PCR on the colonies obtained from the third Gibson.
28 September
- Gel electrophoresis on the colonies obtained from the third Gibson. Observation: No results.
- Cultivation of U6, RBCS2 terminator, pLIP and LIP-RFP.
- PCR on the colonies obtained from the third Gibson.
29 September
- Gel electrophoresis on the PCR product from the previous day. Observation: The bands indicate that there is no insert.
1 October
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. Observation: Correct bands were obtained!.
- Plasmid preparation of pLIP, LIP-RFP and RBCS2 terminator.
- New cultivation of pLIP on plates.
2 October
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. Observation: Correct bands were obtained!
Week 17
3 October
- Sequencing of pLIP, LIP-RFP and RBCS2 terminator.
5 October
- New Gibson Assembly on pSB1C3 with U6.
- Gibson transformation on pSB1C3 with U6.
6 October
- PCR on U6.
- Cultivation of LIP-RFP.
7 October
- Gel electrophoresis on U6. Observation: The band indicate that there is no insert.
- Cultivation of algae.
8 October
- Cultivation of LIP-RFP.
9 October
- Plasmid preparation of LIP-RFP.
Week 18
11 October
- Plasmid preparation of pLIP and RBCS2 terminator.
12 October
- Gel electrophoresis on pLIP, LIP-RFP and RBCS2 terminator. This was the last day at the lab!
Week 19
19 October
- Parts were finally prepared for submission!
20 October - Parts submitted
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