Team:Linkoping Sweden/Experiments

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 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 (BBa_K2095000), U6 promoter, RBCS2 terminator (K2095002) and LIP-RFP (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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