Team:Wageningen UR/Notebook/Sophie
Here you can find the notebook from Sophie.
Wetlab - May
- Week 1: 10th of May - 14th of May
- Week 2: 17th of May - 21th of May
- Week 3: 24th of May - 28th of May
- Week 4: 31st of May - 4th of June
Introduction into the laboratory. Lab safety, best practices and etc. Waited for arrival pTR47m4-GFP and gblocks.
PCRs of lacI, Prha. Waited for pTR47m4-GFP and gblocks.
PCRs of hok, sok, hybrid promoter. Waited for pTR47m4-GFP and gblocks.
Isolated pTR47m4-GFP from bacterial stab, verified by sequencing. PCRs of FrmR, terminators, Pfrm, backbone.
Wetlab - June
- Week 5: 7th of June - 11th of June
- Week 6: 14th of June - 18th of June
- Week 7: 21st of June - 25th of June
- Week 8: 28th of June - 2nd of July
Redid some PCRs to obtain better yields. Tried first Gibson assembly with fragments 1-10, no colonies.
Repeat some PCRs for better quality fragments. New Gibson assembly with fragments 1-10, several colonies observed, but cPCR gave no bands for insert and sequencing confirmed this.
No labwork done.
Designed alternative Gibson approaches. Redid PCRs with new primers for different overhangs.
Wetlab - July
- Week 9: 5th of July - 9th of July
- Week 10: 12th of July - 16th of July
- Week 11: 19th of July - 23th of July
- Week 12: 26th of July - 30th of July
No labwork done.
Redid some PCRs to obtain better yields. Tried Gibson assembly with fragments 1-5. Few colonies, and cPCR confirmed no inserts. Tried Gibson assembly with fragments 1-5 with different ratio's. Few colonies, and cPCR confirmed no inserts.
Tried new Gibson assembly with fragments 1-,5,10. Few colonies, cPCR and sequencing proved no insert. Redid some PCRs for fragments 1,3. Started designing first formaldehyde toxicity test.
Tried new Gibson assembly with fragments 1-5,10 in different ratio's. Colonies were observed, but cPCR proved again no insert. Tried Gibson assembly with fragments 3,4,5. This time, many colonies. cPCR and sequencing proved correct insert of Prha and FrmR in plasmid backbone. Continued determination of formaldehyde concentrations for toxicity test.
Wetlab - August
- Week 13: 2nd of August - 6th of August
- Week 14: 9th of August - 13th of August
- Week 15: 16th of August - 20th of August
- Week 16: 23th of August - 27th of August
- Week 17: 30th of August - 3th of September
Glycerol stock of colonies with pSafety_A_FrmR (3,4,5) and linearized plasmid as new backbone. Tried new Gibson assembly with [3,4,5],1,2,10. Few colonies and cPCR confirmed no insert in backbone. Obtained BW25113, SIJ488 wildtype and SIJ488 ΔFrmRAB strains for toxicity test and made glycerol stocks.
Redid some PCRs for 7b and 2 with overhang primers to ligate 1,2,10 and attach 3' end of hybrid promoter to GFP gene. Tried Gibson assembly with [1,2,10] and [3,4,5], which failed. Did overlap extension PCR for 1,2,10 and tried same Gibson assembly again, but also failed. Sequenced PCR product [1,2,10] seemed to be correct. Did overlap extension PCR for fragments 6,7,8,9 and 6,7 and 8,9 separately. Obtained KEIO strain FrmR and verified knock-out by cPCR, but used wrong primers.
Redid PCRs for 6,7 and 8,9 and GFP, low yields. Tried Gibson assembly with different combinations of [3,4,5] and 6,7,8,9 and [6,7] and [8,9]. Few colonies, cPCR showed a few bands at wrong height. Sequencing showed only [7b,8] was in, 3 disappeared. New cPCR for BW25113 ΔFrmR confirmation, but not verified yet. Made pre-cultures for toxicity test, and started first test.
No labork done.
Verified BW25113 ΔFrmR. Redid some (overhang) PCRs for plasmid fragments to obtain better yields. Analysis of first toxicity test results: blanc was contaminated so results not used.
Wetlab - September
- Week 18: 6th of September - 10th of September
- Week 19: 13th of September - 17th of September
- Week 20: 20th of September - 24th of September
- Week 21: 27th of September - 1st of October
Redid many (overhang) PCRs for plasmid fragments with new primers to obtain better yields and right overhangs. Sequenced backbone to check Prha if was still in there. Ordered gblock lacI module.
Redid many PCRs for plasmid fragments with new primers to obtain better yields and right overhangs. Tried linear Gibson assembly with fragments 6-9 and used directly for PCR. Gave no bands on gel.
LacI gblock confirmed not to be present, ordered new. Redid PCRs for plasmid fragments. Tried linear Gibson for fragments 7-9 and used for PCR. Gave no bands.Tried linear Gibson for fragments [10,1,2],5 and used for PCR. Gave no bands. Obtained [7,8]. Prepared new toxicity test.
Redid toxicity test. Blanks again contaminated. Tried obtaining LacI module and [7,8] with different PCR strategies, but did not work. Prepared growth experiment SM1 strain.
Wetlab - October
- Week 22: 4th of October - 8th of October
Redid toxicity test twice, with mineral oil added: no contaminations. Also did toxicity test for SM1 strain. Analysed results and made growth curves.