Team:Linkoping Sweden/Experiments

Overview on Laboration

Week 1: 13 – 19 June

14 June: First day at the lab! Making Hutner’s trace elements

Week 2: 20 – 26 June

21 June: Making SOC-medium, LB-medium, LB-agar and Chloramphenicol plates. 46 agar plates were made.

Week 3: 27 June – 3 July

27 June: Transformation of E1010 - The transformation was successful 28 June: Control of competent cells 29 June: Transformation of E1010 to super competent XL-1 - The transformation was successful. 30 June: Making E.Coli Calcium Chloride competent cells 1 July: - Making solutions for TAP- and TRIS medium - Cultivation of XL1 and E1010

Week 4: 4 – 10 July

4 July: - Making LB-medium and LB-agar. - Plasmid preparation of E1010 - Test cultivation of algae 5 July: - Making agar plates - Digestion and ligation of LIP, U6, UTR and LIP-RFP. - Transformation of E1010 and MD-cells competent test - First algae cultivation 6 July: Transformation on U6, LIP, LIP-RFP and UTR. Colonies for U6 and LIP were detected. 7 July: Cultivation of U6, LIP, LIP-RFP colonies on new plates with Chloramphenicol 8 July: OD measurement of transformated bacteria.

Week 5: 11 – 17 July

11 July: PCR on Cas9 12 July: Gel electrophoresis on Cas9 to see if the PCR succeeded. The gel did not show any bands for Cas9 13 July: PCR 14 July: PCR on pSB1C3 15 July: - Gel electrophoresis on pSB1C3 No bands were obtained on the gel. - PCR on pSB1C3

Week 6: 18 – 24 July

18 July: PCR and gel electrophoresis on pSB1C3 No bands were obtained. 20 July: PCR and gel electrophoresis on pSB1C3 We obtained bands on the gel at approximately 2000 bp. 22 July: Digestion and ligation on LIP, U6, UTR, Cas9, LIP-RFP, sgRNA and pSB1C3.

Week 7: 25 – 31 July

25 July: - PCR on LIP and UTR from colonies - Cultivation of LIP and UTR colonies on new plates - PCR purification 26 July: - Gel electrophoresis on pSB1C3, UTR and LIP No bands were obtained. - Digestion and Ligation on LIP-RFP and pSB1C3. 27 July: New project approach Transformation of LIP-RFP and pSB1C3.

Week 8: 1 – 7 August

1 August: - Cultivation of Hyg - Gel electrophoresis on UTR and LIP Bands were obtained at 700 bp. 3 August: - Plasmid preparation of LIP, UTR and Hyg. Was later show to be wrong - Transformation of LIP-RFP, U6, sgRNA and Cas9. 4 colonies on sgRNA and 4 colonies on LIP-RFP. No colonies on Cas9 and U6. 4 August: Making TAP medium for cultivation of algae in the dark

Week 9: 8 – 14 August

8 August: - PCR on LIP-RFP and sgRNA - Cultivation of LIP-RFP and sgRNA colonies on new plates - First algae cultivation in darkness 9 August: - Transformation of U6 and Cas9. - Gel electrophoresis on LIP-RFP and sgRNA. No bands on the gel. 10 August: - PCR on LIP-RFP and sgRNA - Gel electrophoresis on LIP-RFP 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. Bands at 300 bp were obtained. sgRNA with primers should be at 200 bp. 12 August: Gel electrophoresis on Cas9 and U6. Cas9 showed no bands. U6 showed bands at 350 bp. U6 with primers should be at 515 bp.

Week 10: 15 – 21 August

15 August: - Preparation of TAP agar Because of difficulties with the gas no plates could be performed today. - Gel electrophoresis on UTR, LIP, Hyg and pSB1C3. pSB1C3 showed at 2000 bp as it was supposed to. The other fragments did not show any bands. - Cultivation of Hyg. 16 August: - Cultivation of sgRNA, LIP-RFP and U6. - PCR on Cas9 and Hyg - Gel electrophoresis on Cas9 and Hyg The gel showed a weak band on Cas9 around 4000 bp. 17 August: Plasmid preparation on LIP-RFP, U6 and sgRNA Was later show to be wrong 18 August: - Cultivation of algae for transformation It took 5 days for the algae wild type to reach OD 1,757 The mutant alga evaporated - Making TAP agar plates - Making TAP Hygromycin plates

Week 11: 22 – 28 August

22 August: Cultivation of LIP-RFP and Hyg 23 August: - Plasmid preparation on LIP-RFP and Hyg - PCR on Cas9 and pSB1C3 - New cultivation of algae in the dark 24 August: - PCR on LIP-RFP, Hyg and pSB1C3 - Gel electrophoresis on Cas9, Hyg, LIP-RFP and pSB1C3. Bands for Cas9 and Hyg were obtained. - PCR purification of Cas9. 25 August: - Gel electrophoresis on pSB1C3 Bands were detected at 2000 bp which match with pSB1C3 - PCR purification on pSB1C3 - First Gibson Assembly! - Transformation of Gibson Assembly Colonies were obtained! - PCR on Cas9, Hyg, pSB1C3 26 August: - PCR on Gibson Assembly product and colonies from Gibson Assembly transformation - Chloramphenicol plates 52 plates were made! - Gel electrophoresis on PCR product from Gibson Assembly, Cas9, Hyg and pSB1C3 Bands were detected for all the DNAs!

Week 12: 29 August – 4 September

29 August: - Gel electrophoresis on Gibson Assembly colonies A band at 5500 bp was obtained. We want bands at 7000 bp. - Digestion on LIP-RFP - PCR on Gibson Assembly colonies. 30 August: - PCR on Gibson Assembly colonies. - Cultivation of Gibson Assembly coloni on new plates - Ligation on LIP-RFP with pSB1C3. - New Gibson Assembly transformation 31 August: - Gel electrophoresis on Gibson Assembly colonies No bands. - Plasmid preparation on Gibson Assembly colony. 1 September: - PCR on plasmid prepared U6, UTR, LIP, LIP-RFP, sgRNA and Hyg. - Gel electrophoresis on LIP, sgRNA, U6, UTR, LIP-RFP and Hyg. No result on the gel. 2 September - Second Gibson assembly - Gibson transformation - Transformation LIP-RFP 3 September - PCR and gel electrophoresis on gibson colonies No results - Digestion and ligation of LIP, U6, UTR, sgRNA and pSB1C3 4 September - PCR and gel electrophoresis on gibson colonies It looks like Colony 8 has a band at 7000 bp! Yeeey!

Week 13: 5 – 11 September

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 Hyg in the LB-media Screening of colonies from Gibson Assembly Bands were obtained, but no band was at 7000 bp. 7 September PCR and gel electrophoresis on Hyg 8 September Plasmid preparation of Gibson Assembly colony 8 Screening on Gibson colonies 9 September The sequences were obtained We did not insert Hyg but instead YFP was inserted. Cas9 and LIP are inserted successfully! 10 September Cultivation of algae mutants and Gibson colony 8 Gel electrophoresis on plasmid preparation of Gibson colony 8 and Gibson colonies The plasmid preparation of Gibson colony 8 showed good bands. 11 September PCR on some Gibson colonies Preparation for plasmid preparation The cells were centrifuged and the pellet was saved for later continuation of plasmid preparation Cultivation of Gibson Assembly colonies on new plates

Week 14: 12 – 18 September

13 September OD measurments on the algae Gel electrophoresis on the PCR product from 11/9 - 16. Band on 300 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp 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 16 September Electroporation on algae The algae have grown well. 17 September PCR on Gibson colonies Continuation on the electroporation from previous day. 18 September Gel electrophoresis on Gibson colonies

Week 15: 19 – 25 September

19 September Sequenced was obtained Looks like we did not insert U6 and sgRNA :( 21 September PCR on Gibson 3 Gel electrophoresis on the PCR product from today Band were obtained at 300 bp and 2000 bp. 22 September Gel electrophoresis on PCR product from yesterday Bands at 2000 bp and 300 bp were obtained Gibson on pSB1C3 with LIP, LIP-RFP, U6 and UTR respectively. Transformation on all the Gibson product 23 September Gel electrophoresis on Gibson 3 colonies No bands. PCR of Gibson with LIP, LIP-RFP, U6 and Term. Screening of YFP transformed algae No proof that the transformation worked. 24 September Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP Bands were obtained at 500 bp for Term, 600 bp for LIP and 1500 bp for LIP-RFP. No result for U6. Gel electrophoresis on Gibson 3 colonies No bands. PCR on Gibson with U6, Term, LIP and LIP-RFP Cultivation of U6, Term, LIP and LIP-RFP 25 September PCR on Gibson 3 colonies Gel electrophoresis on Gibson with U6, Term, LIP and LIP-RFP. Bands were obatined. Cultivation of U6, Term, LIP and LIP-RFP

Week 16: 26 – 30 September

26 September PCR on U6 colonies Gel electrophoresis on Gibson 3 colonies No result. Plasmid preparation on LIP, LIP-RFP and Term. 27 September Plasmid preparation nr 2 on LIP, LIP-RFP and Term. Gel electrophoresis on U6 colonies No result. PCR on Gibson 3 colonies 28 September Gel electrophoresis on Gibson 3 colonies No result. Cultivation of U6, Term, LIP and LIP-RFP PCR on Gibson 3 colonies. 3 October Sekvensing of LIP, LIP-RFP and Term

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