16th – Thursday
- Restreaked B. subtilis and B. licheniformis on LB-plates.
- Autoclaved BG-11 media.
- Biobrick BBa_E0240 from DNA Distribution kit 2016 plate 4 spot 11N where transformed.
17th – Friday
- Placed E. coli transformants in 4°C room.
- Prepared 50% DMSO solution.
- Prepared new BG-11 medium for Synechocystis with 50 mM NHCO3 and 25 mM HEPES. pH was adjusted to 7.65.
- Prepared 50% glycerol solution.
- Prepared BG-11 agar plates. 10 without antibiotics + 10 with chloramphenicol.
- Restreaked cyanobacteria plates, WT + JA06, placed on lab bench.
- Prepared TE-buffer and NaCl solutions for DNA extraction from cyanobacteria.
20th – Monday
- Prepared solutions for BG-11 plates with buffer, NaHCO3 and L-arginine.
- Changed the parafilms of the shake.
- Restreaked Synechocystis JA06 from plate "Hudson 7/6" onto plate "JGH 20/6".
- Prepared solutions for genome extraction.
21st – Tuesday
- Made Kanamycin stocks (50 mg/mL).
- Added spectinomycin (20 µg/mL) and kanamycin (25 µg/mL).
- Renew medium for Synechocystis.
- Made 10 µM stocks of AckA F and R.
- Colony PCR with AckA F and Acka R.
- LB+kanamycin (50 µg/mL) plates for E. coli.
- Inoculate culture containing CRISPRi.
- Inoculate colony of B. licheniformis.
22nd – Wednesday
- Miniprep CRISPRi plasmid
- Genomic extraction of B. licheniformis.
- Genomic extraction of Synechocystis.
- Set up equipment for growing of Synechocystis.
- Make BG-11 with 3% acetate, HEPES & NaHCO3.
- Make BG-11 with 3% acetate, uracil (60 mg/L), HEPES & NaHCO3.
23rd – Thursday
- Made a diluted stock of pCyJ2 for PCR's.
- PCR AckA, primers: AckA F + AckA R (64°C, synechocystis genome).
- PCR pTrc, primers: pTrc F (KmR) + pTrc R (AckA) (64°C, pCyJ2).
- PCR pNF (SpR primers).
- Set up lights in 30°C room for Synechosystis liquid cultures.
- Gel electrophoresis to verify products from first PCR.
26th – Sunday
- Overnight cultures of S. cerevisiae in YPD to do the Growth curve.
- Spin down of Synechosystis cultures JA06 3000rpm 6 min.
27th – Monday
- Transfer Synchosystis liquid cultures to fresh media.
- Restreak B. licheniformis and B. subtilis.
- Start S. cerevisiae acetate growth trials in BG-11 medium.
- Run gel for second Synechosystis PCR.
- Redo failed PCR.
- PCR Synechocystis constructs.
- Transformation of BBa_K584001.
- Restreak E. coli production strains.
28th – Tuesday
- Set overnight with pKD3.
- Restreak WT Synechocystis on BG-11 plates without antibiotics.
- Inoculate CRISPRi colony overnight, starting at 17:00 (use spectinomycin (50 µg/mL) instead of amp).
- Set overnight culture with BBa_K584001.
- Make LB+ spectinomycin plate.s
- Inoculate Synehocystis colony in BG-11 to receive new liquid culture.
- Dilute all arrived primers.
29th – Wednesday
- Miniprep of BBa_K584001.
- Miniprep PL-22 dCas9 plasmid.
- Miniprep of pKD3.
- PCR Synechosystis.
- Yarrowia genomic extraction from colony.
- Run pTrc and tB0015 on a gel (use 85V 45 min).
- Transform CRISPRi plasmids to E. coli: PL22-sgRNA-NTI (kanamycin) and psbA1-psbA2-dCas9 (spectinomycin).
30th – Thursday
- Gibson assembly of synechosysts 1.1.
- PCR of gibson product synechosystis 1.1.
- Fusion PCR of synechosystis 1.1 fragments.
- Transformation of E. coli for plasmid miniprep.
- BBa_E0840 and BBa_K283003 transformation.
1st – Friday
- Miniprep PL22-sgRNA-NTi and psbA1-psbA2-dCas9 (grab colony from plate).
- Inoculate new Synechosystis colonies in liquid BG-11.
- PCR of synechosystis 1.6 HO glnA Dw and 1.7 HO acs Dw.
- PCR: E.Coli, Yarrowia, Cerevisiae.
- Gibson of Syn. 1.2 and 1.3.
3rd – Sunday
- Overnight culture PL22-sgRNA-NTi.
- Overnight culture psbA1-psbA2-dCas9.
- Overnight culture p416.
- Overnight culture E.coli production strain.
4th – Monday
- Sort the diluted primers.
- Take out PCR tubes from machine. Run on gel.
- Purify PCR products (glyoxy, GIbson 1.1, 1.6 HO glnA Dw, 1.7 HO acs Dw).
- Transform BBa_E0840 and BBa_K823003 with HIGH competence cells.
- Miniprep PL22-sgRNA-NTi.
- Miniprep psbA1-psbA2-dCas9.
- PCRs as many as possible.
- PCR of 1.4 pta + HO acs Dw (in freezer, add polymerase).
- Make inventory of 4°C room.
5th – Tuesday
- Make BG-11 plates with kanamycin and spectinomycin.
- Set overnight cultures for genomic DNA extraction/plasmid miniprep, 5 ml LB-medium w/ appropriate antiobiotics 37°C shaker:
- B.Subtilis production strain
- B.Subtilis WT
- DH5a HEME A-D HEME C-A C15 plasmid
- PCR E.coli, 15s extension time, plasmid template.
- PCR Yarrowia/Saccharomyces, 15s extension time, genomic DNA template.
- PCR Yarrowia/Saccharomyces, 17s extension time, genomic DNA template.
- Gibson 1.5 1.6 1.7 PCR.
- Restreak Synechocystis & suspend Synechocystis from plates in BG-11.
- Overnight cultures:
- pKD3 (LB + amp)
- BBa_K823003 (LB + chloramphenicol)
- BBa_E0840 (LB + chlorampenicol)
- p416TEF (LB + amp)
- DH5a (E. coli prod. strain) (LB + amp+sepctinomycin)
- B. subtilis WT (LB)
- B. subtilis 168 trp+ (LB)
6th – Wednesday
- Miniprep pKD3
- Miniprep BBa_K823003
- Miniprep BBa_E0840
- Genome extraction:
- DH5a (E. coli prod. strain)
- B. subtilis WT
- B. subtilis 168 trp+
- Run gel for:
- 1.5 Syn. Gibson
- 1.2 glnAUp+kmR
- 1.2 AckA+glnADw
- 4.1 tLIP2
- 5.6 pPYK2
- PCRs for S. cerevisiae promoter study.
- Restrict and ligate BBa_K823003 with BBa_E0840.
7th – Thursday
- Rerun BBa K823003 with restriction enzymes EcoR1 & Spe1.
- Ligate BBa K823003 & BBa E0840 and verify ligation product.
- Measure concentrations of genomic extractions from 6/7 also diluted them.
- PCR and gel of 4.1 and 4.3 tXPR2 and 5.11 pPYK2.
- PCR and gel of 3.3 ahrC Dw 3.3 ahrC Up and 2.1 FRT.
- Make autoclaved MQ.
- Autoclaved lithium acetate 2M.
- Restreak B. subtilis and B. lichenformis plates.
- Gibson of 1.2.
8th – Friday
- Make Gibson of 1.3 with newly purified 1.3 RFP.
- GEL PCR for 1.2 gibson PCR product on gel. Tubes labeled 60, 61, 62, 63, 64, 65, 66.
- GEL PCR for 1.5 Gibson, GFP PVEG, 5.4 pGLN1, 5.5 pPCK1, 5.8 pFBT(HO pAQR1), 5.10 pPCK1(pGLN1).
11th – Monday
- PCR purification 1.2 syn.
- Run Gibson product 1.3 and PCR prod. 5.11 on gel.
- PCR purification kit of GFP pVeg, 5.4 pGLN1 and 1.5 Gibson product.
- Restreak all plates.
- Make new Synechocystis liquid cultures (with and without chloramphenicol).
- Autoclave BG-11 media for Cerevisiae growth tria.l
- Rewrap Synechocystis plates with fresh parafilm to avoid drying. Two older plates were thrown away, they were not healthy.
12th – Tuesday
- Run gel verification for the 6 PCR’s run on the 1.2 construct.
- PCR for 4.1 Gln1, 4.3 AQR2, and 5.11.
- PCR purification of Synechosystis 1.1, 4.1 Glu 1, 4.2 PTef, 4.2 pTEF1, 4.3 AQR1, 5.11 pPYK2.
- Finish the plate for BG-11 and BG-11 with Chloramphenicol (25µg/ml).
- Make diluted Chloramphenicol solution (25mg/ml).
- Run PCR again on purified PCR product 1.1 with 1,5 argH Up F (IS) and 1,5 argH Dw R (IS).
- Restreak plates.
- Make new liquid cultures for Synechocystis.
13th – Wednesday
- Make LB plates with 25 µg/ml Chloramphenicol.
- Run 1.1 Syn. Gib. and 1.2 Syn. Gib. on gel.
- Run the rediscovered PCR for E. coli.
- PCR of Gibson 1.5.
- PCR of 5.8 pFBP1 (HO pAQR1).
- Continue by cutting pNF plasmid with SphI and XhoI (results in 2 bands: 3349 and 2648).
- Purify 2648 band from gel (this plasmid can be used 1.1 as well).
- Media preparation (MMx10, subtilis salts) for the B.subtilis transformation.
- Change air in liquid 1 ml synechosystis cultures.
- Rewrap synechosystis plates with fresh parafilm.
14th – Thursday
- FRTcmr gradient between 61-63. If failed try with Primestar (anneal at 55°C).
- #1 Arg_mut megaprimer PCR (try 6s extension time and gradient from 56°C to 64°C).
- Repair parafilm on Synechocystis plates.
- Make other required solutions, such as glucose 50% & tryptophan 1%.
- Set overnight cultures for cryostocks (5 Yarrowia strains).
- Run gel for purification of 1.4 pta + HO acs Dw.
15th – Friday
- Finish the M1 and M2 solution by mixing with glucose, tryptophane.
- Gel verification and purification of 1.4 pta + HO acs Dw.
- Run Gibson reactions for 1.1 1.2 1.3 1.4 with long incubation times.
- Genome extraction with the MG1655 and the TOP10 strains.
- Verify the two purified FRTcmR products on a post-stain gel.
- Gibson of yarrowia 4.1 construct.
18th – Monday
- Purify the GEL product of ArgMut #1.
- Run ArgMut#2 PCR.
- Try the Gibson for 4.1 two/three at a time (inc for 2h.)
- Try WT Synechocystis on glucose media.
- Set overnight cultures for E.coli and B. subtilis for cryostocks.
- Start new Synechocystis liquid culture with glucose.
19th – Tuesday
- Try PCR for ArgMut second reaction in temperature gradient with longer extension time(20sec).
- Miniprep B. subtilis plasmids from plate colonies.
- Cryostocks for overnight cultures of B. subtilis and E. coli production strains.
- PCR’s with new primers (Syn 1.1, 1,2).
- Set overnight culture of pBS1C and pBS4S (B.subtilis plasmids) in E. coli for miniprep.
- Measure pH of BG-11 media.
- PCR 3.2 AmyE+cmr+AmyE and 3.3 spc casette.
20th – Wednesday
- Ligate glyoxylate shunt into plasmid:
- Cut pBS1C with XbaI, SpeI (BcuI) and FastAP (prevents recircularization) 15 min 37°C.
- Cut purified 3.1 glyoxylate shunt with XbaI and SpeI (BcuI) 15 min 37°C.
- Purify both using PCR clean kit (SpeI is not inactivated by heat).
- Measure concentration.
- Prepare RF15 yeast strain for making competent stock → start overnight culture.
- PCR of p416-TEF1-GFP.
- Overnight liquid culture of E.coli with p416-TEF1, pMel- 10, PNF delta phaA::KmR.
- Miniprep of B.subtilis plasmids pBs4S and pBs1C from liquid cultures.
- Restreak Synechocystis.
- Autoclave tubing and filters for air flow for Synechocystis liquid culture.
- Start liquid Synechocystis culture with air flow.
- Exchange air in Synechosystis liquid cultures.
- Throw away failed PCR’s from the unpurified samples box.
- PCR 5.1 p416 (GFP plasmid without promoter, linearized).
- Miniprep p416-TEF1-GFP (from plate) → gave 164 ng/ml.
- Transform yeast with p416-TEF1-GFP.
21st – Thursday
- Make stock for NaHCO3.
- Start two WT Synechocystis liquid cultures in shake flasks, use ~10-20 ml media (one with standard BG-11 & one with BG-11 + new NaHCO3 mix) , take a lot of cells from plates to suspend in liquid (do not take only 1 colony but scrape up at least 2-3 “large loops”). Keep cultures on shaker @ 120 rpm (optimally we want them on shaker to the right in 30°C room which currently is occupied shaking @ 200 rpm).
- Amplify the 2,1 Gibson in different template amount and temperature gradient(60°C,62°C).
- Redo the PCR for 1.2 DO AckA with temperature gradient and different extension time.
- Run the gel for 4.1 (1-2) and 4.1(3-5).
- Rerun PCR of pPCK1 HO.
- Miniprep of p416-TEF1, pMel-10, PNF delta phaA::KmR.
22nd – Friday
- Book shakers for growth trials next week.
- Take care of yeast transformed with p416-TEF1-GFP → Put into 4°C room.
- Start new liquid Synechocystis cuture with WT in new correct BG-11 + HEPES + NaHCO3.
- Gel for pBS1C+Glyoxylate PCR product: labeled as (A): 6326bp → freezer.
- Do something with p416-TEF1-GFP → Primestar PCR protocol.
- Start with pMel-10 → PCR:d it. Gel tomorrow.
- Gel for PCR of pPCK1 HO from yesterday.
- Finish the BG-11 + HEPES from thursday 21/7.
- Add NaHCO3 (22,22ml) to the BG-11 + HEPES via filter sterilization.
- Throw out old Synechocystis liquid cultures.
- Book Fluostar Omega for fluorescence measurement.
24th – Sunday
- Inoculate Yarrowia JMY2101 (Ura-, Leu+), S. cerevisiae production strain, S. cerevisiae nonproducing strain.
- Add pBS1C+Glyoxy isolated colonies (1 to 8) to LB+Amp media tubes to miniprep on monday.
25th – Monday
- Start growth trial.
- Purify 1.1 HO argH UP(IS).
- Try PCR 1.1 HO argH DW (IS) again.
- Miniprep on E.coli colonies with glyoxylate shunt.
- Make gel for the pMel-10 PCR that was carried out 22/7. → Gel shows PCR was successful.
- Figure out what to do wtih the p416-TEF1-GFP and the pPCK1 HO → PCR of p416-TEF1-GFP.
- Inoculate transformed yeast for fluorescence measurement. Fluostar booked 14.00-16.00 on tuesday and wednesday. Don’t forget blank! --> Made overnight in YPD.
- Autoclave things for liquid cultures with air flow.
- Start new liquid Synechocystis culture.
26th – Tuesday
- Synechosystis DNA extraction from liquid culture.
- Fusion PCR for 2.1.
- Fusion PCR for 3 SpR casette, 4 SpR casette and 7 Spr casette.
- Make SD -ura for promoter study.
- PCR purification of pMel-10 PCR product.
- Gel for p416-TEF1-GFP and then probably rerun it.
- Grow yeast with p416-TEF1-GFP to appropriate OD and measure fluorescence.
- Synechocystis liquid culture updates.
27th – Wednesday
- Gel pic for 1.3 and 1.4 SpR cassette
- Do the remaining syn 1.1 - 1.4 PCRs. Use 1 µL of diluted genomic DNA as template. Print the updated excel PCR sheet.
- Run the restriction verification reactions of the shunt again on post-stain GelRed gel.
- Check in microscope for contaminations.
- Restreak colonies of transformed S.cerevisiae with p416 TEF1 gfp on SD -ura plate. Also restreak cas9Δura.
- Gel for the p416-TEF-GFP PCR gradient with DMSO.
- Check on the SD -ura media to see if there is any contamination.
- Dilute air flow culture of Synechosystis (25 µg/ml chloramphenicol).
28th – Thursday
- Finalize M1 and M2 solution for B.subtilis transformation and Stock1 solution for BG-11.
- Make BG-11 + Spectinomycin + chloramphenicol + L- arginine plates.
- Start the PCRs for 1.1 HO argH UP, DW/ 1.2 HO glnA UP,Dw / 1.3 HO acs UP,Dw.
- Try 1.4 pta with gradient.
- Purify Spr cassette and mRFP1.
- PCR extraction of GFP from p416-TEF1-GFP.
- PCR of p416-TEF1-GFP with new primer.
- Check restreaked plates from yesterday.
- New Synechosystis JA06 liquid culture in BG-11 + NaHCO3.
- Dilute Synechosystis liquid cultures.
29th – Friday
- Restreak B.subtilis and B.licheniformis on LB plates.
- Take out plate from Aida (with pBS2E) from 37°C oven.
- Miniprep JME1047 and JMP2563.
- Check 5.11 pPYK2 (HO pGLN1) on gel again.
- Transform yeast with p416-TEF1.
- Try 1.4 pta with lower template amount/DMSO.
- Gel PCR 1.1 HO argH DW(1045).
- Verification of pPYK2 on gel.
1st – Monday
- Purify GFP.
- Check 4.3 AQR1 on gel again.
- Purify 5.10 pPCK1 (HO GLN1) and check on gel.
- Set cultures of Yarrowia JMY195 to make competent cell stock.
- Wrap synechosystis plates with new parafilm.
- Restreak Yarrowia plates.
- Linearize JMP2563.
- Synechocystis update.
- Restreak E.Coli containing the ligation of Glyoxilate shunt + plasmid.
- PCR to check the construct on pBS1C.
2nd – Tuesday
- Assembly of 4.3 Yarrowia construct + transformation to E.coli.
- Start cleaning and sorting out the unpurified PCR products.
- Try PCR for 1.1, 1.2, 1.3 for UPs with Phusion with DMSO.
- Take a picture of GEL for 1.4 acs DW, 1.2 glnA UP, 1.2 glnA DW.
- Gibson of pAQR1 construct.
- Start PCRing BioBricks.
- Make competent cell stock of Yarrowia JMY195 (Po1d).
- Miniprep of the pBS1C+Glyoxy ligation.
- Linearize pNF(KmR) with Sphl(Pael) and Xhol.
3rd – Wednesday
- Check 4.3 Gibson transformants, replate 8 single colonies from 2 µL or 5 µL plate on LB + Kan and inoculate in 3mL liquid LB + Kan overnight.
- Prepare Gibson reaction for syn 1.2.
- Linearize pNF (KmR) with XhoI and PaeI.
- Make SD -URA -LEU plates solution #2 for second Yarrowia transformation.
- Run BB-pPCK1 on gel for verification.
- Cut BB-pAQR1 and vector (in plasmid box marked with BB RFP on lid) according to biobrick protocols.
- PCR purification of cut BB-pAQR1 & BB-pPCK1 pcr product.
- PCR BB-pPYK2.
- Throw out some old liquid Synechocystis cultures.
4th – Thursday
- Prepare and autoclave agar solution for SD -URA -LEU plates.
- Miniprep 4S colony with the shunt (B.subtilis)
- Miniprep JMP2563 with 4.3 Gibson insert from overnight cultures.
- Miniprep plasmid from colonies with 5.2 (p416-pAQR1-GFP).
- Next promoter study gibson. → PCK1 and pPYK2.
- Colony streak of the promoter study gibson from yesterday i.e. pFBP1 and pGLN1.
- GEL PIC for PCR 1.1 arg UP 1.3 acs UP.
- Synechocystis update, dilutions etc.
5th – Friday
- Make plates: BG-11 + kanamycin(25µg/ml) + L- glutamine (5 mM) -> 0.7307 g/L, needed for 1.2 transformation).
- Miniprep for 1.2 Synechocystis.
- Restriction verification of Syn 1.2
- Check pPCK1 and pPYK2 transformants and colony streak + inoculation overnight liquid.
- Miniprep, restriction and verification of pFBP1 and pGLN1.
- B. Subtilis transformation of S4: pBS4S + Glyoxy shunt
- p416-pAQR1-GFP verification
- Gel purification of BBpSB1 and pNF (Kmr)
6th – Saturday
- Miniprep, restriction and verification of p416-(pPCK/pPYK)-GFP.
8th – Monday
- Figure out whlat to do with pFBP for S. cerevisiae.
- Transform S. cerevisiae with the working p416-promoter-GFP. (all the working 416-xxx-GFP plasmids, ie pAQR1, pGLN1, pPCK1 and pPYK2)
- Restriction verification of 1.2.
- Gibson 1.1 Synechocystis.
- Restreak plates of B.subtilis and B.lichenformis.
- Made new SD -LEU plates.
- Check fragments for 2.1 E.coli construct.
- Linearization of 4.3 Yarrowia construct.
9th – Tuesday
- Cut and verify p416-pFBP with new enzymes.
- PCR of 5.3 pFBP1 (p416).
- Cut and verify 1.2 Syn with new set of enzymes.
- Transformation of 1.1 Synechocystis constructs.
- Additional gel to control the 2.1 E.coli construct fragment.
- Gel purification pNF(cut with SphI and XhoI) and pSB1C3(cut with XbaI and PstI).
- Ligation and transformation of BB pSB1C3+pAQR1.
- Verification gel of PCR BB pPYK2.
- Yarrowia Transformation with 4.3.
10th – Wednesday
- Verification of Kmr cassette and 1.2DO gln Dw.
- Restreaked and inoculated 1.1 Synechocystis plates.
- Gel of pFBP1 from PCR.
11th – Thursday
- Miniprep for 1.1 Synechocystis
- Miniprep BBpAQR1 colonies
- Restriction verify the BBpAQR1
- Gibson of and transformation with pFBP
- Check plates with yeast transformations of p416-(pAQR/pGLN/pPCK/pPYK)-GFP
- Cut and verify 1.2 Synechocystis with new set of enzymes
- Yarrowia 4.3 Transformation
- Bacillus subtilis growth trial with threonine in BG11
- 4.1 tLIP2 (DO) Primestart PCR
- 4.1 GFP (DO) Phusion PCR
12th – Friday
- Restriction verify with BcuI and PaeI of 1.1 Synechocystis.
- Phusion PCR 4.1 pTef(DO).
- Clean out unwanted Saccharomyces cerevisiae plates.
- Colony streak p416-pFBP-GFP.
- Phusion PCR of BB-pAckA.
- Transformation of BB-pPYK1.
- Ligation of BB-pPCK1.
- Transformation of BB-pPCK1.
- Gibson of 2.1 E.coli construct.
14th – Sunday
- Setting overnight liquid cultures of p416-pFBP-GFP.
15th – Monday
- Miniprep, restrict and verify p416-pFBP-GFP.
- Genomic extraction of 4.3 Yarrowia transformant.
- Synechocystis update & care.
- Restriction of JMP1047 plasmid with BamHI and AvrII.
- Restreaking of transformed BBpPYK1.
- Restreaking of transformed BBpPCK1.
16th – Tuesday
- Fluorescence microscope introduction.
- Restriction verification of BBpPCK1 and BBpPYK1.
- Miniprep of BBpPCK1 and BBpPYK1.
- Transformation of E. coli with 4.1 Gibson.
- Repetition of Phusion PCR of BB AckA.
- 1.2 Gibson transformation.
17th – Wednesday
- Synechocystis growth trial.
- Air flow liquid culture autoclavations.
- Cryostock Synechocystis.
- 1.2 Gibson transformation Miniprep.
- PCR BB AckA + 1.6 glnA Dw (DO).
- BB AckA purification.
- PCR 1.6 glnA Dw (DO) - PrimerSTAR.
- PCR of 1.7 with new overhang.
- 1.2 Synechocystis Construct.
- 4.1 Yarrowia construct.
- Inoculation of Saccharomyces cerevisiare with p416-XXX-GFP for promoter study.
18th – Thursday
- OD & HPLC measurements for Synechocystis.
- Restriction of pSB1C3 (BBa_J04450).
- Restriction of BB Acka.
- Promoter study in SD-ura glucose.
- PrimeSTAR PCR of AckA, 1.6 and 1.7.
19th – Friday
- Take out PCR product of pNF from the machine.
- Colony PCR of yarrowia 4.3 with DreamTaq polymerase.
- Ligation of BB AckA and pSB1C3.
- Purification of PrimeStar PCR products AckA, 1.6 and 1.7 (DO).
- Restriction of JMP 1047 with BamHI and AVRJJ (XmaJI).
- PCR pNF 1.8 with gradient
- AckA Transformations in High Competent cells
21st – Sunday
- Inoculation of overnight p416-xxx-GFP for acetate promoter study.
22nd – Monday
- Gibson 1.6 and 1.7.
- Restreak colonies from BB AckA transformed plates. Also inoculate liquid cultures for miniprep.
- Promoter study on acetate.
- Make LB+Kanamycin (50μg/ml) plates
- Cryostock Saccharomyces cerevisiae with p416-XXX-GFP.
23rd – Tuesday
- Miniprepped BB AckA colonies from day before.
- Restriction verify BB AckA plasmids.
- Transform 1.6, 1.7 Synechocystis to E.coli.
- Make our own LB liquid media.
- Make LB + chloramphenicol plates.
- Trial of quantifying Synechocystis based on chlorophyll in the plate reader.
- Promoter study again.
- Promoter study acetate.
24th – Wednesday
- Gibson for 1.4 Synechocystis.
- Prepare all BioBricks for sequencing and shipping.
- Run colony PCR product of yarrowia 4.3 on gel.
- Set overnight culture of JMY330 (Po1d URA+ LEU- ) for competent cell stock.
- Set Synechocystis cultures for transformation.
- Restreak of p416-XXX-GFP and p416-TEF.
- Restreak and inoculate 1.6 and 1.7 Synechocystis plates.
- 4.1 Gibson using 3.3 (JMP1047) plasmid.
25th – Thursday
- Transform 4.1 to E.coli.
- Verify 1.6 and 1.7 by restriction.
- Remake of promoter studies.
- Promoter study samples were sent to sequence.
- Dream TAQ colony PCR.
26th – Friday
- Make BG-11 + kanamycin + spectinomycin + glutamine plates for double knockouts.
- Make LB+kanamycin plates.
- Do gibson of 1.2 with PCR:ed pNF plasmid.
- Redo PCR Acs Up.
- Restriction verification of 1.4 grown on spectinomycin.
27th – Saturday
- GEL pic 4.1 cPCR Yarrowia.
- Transform 1.6 to BG11+ Kanamycin+ L-glutamine plates.
28th – Sunday
- Inoculated and restreaked 1.2 and 1.4 Synechocistis.
- Gibson 1.7 Synechocystis.
- Transformation of 1.7 Synechosystis to E.coli.
29th – Monday
- Sorting and restreaking Saccharomyces cerevisiae plates.
- Transformatin of Yarrowia with p425 and p416.
- Miniprep 1.2 and 1.4 Synechocystis.
- 1.2 Verification.
30th – Tuesday
- Miniprep 1.7.
- Restriction verification of 1.7 Synechocystis.
31st – Wednesday
- 1.7 restriction verification.
1st – Thursday
- Re-do 1.4 from scratch.
- Dilute JMP1047 and PCR.
- Send samples of 1.2 for sequencing.
- Transformation of 1.4 to E.Coli.
2nd – Friday
- Diluted JMP1047 and PCR
- Restreaked and inoculated 1.4 Synechocystis.
- Diluted JMP1047 F and JMP1047 R to 10μM.
3rd – Saturday
- 1.4 Synechocystis plasmid extraction.
4th – Sunday
- Primestar PCR for JMP1047.
- Restriction Verification of 1.4 Synechocystis.
6th – Tuesday
- Gel PCR Product of JMP1047.
7th – Tuesday
- PCR of JMP1047 with Primestar.
- Transformation of 1.7 in Synechocystis.
8th – Thursday
- PCR of JMP1047 verification.
9th – Friday
- Ran and purify JMP1047 from gel
10th – Saturday
- Gibson 4.1 with new PCRed JMP1047.
12th – Monday
- Transformation of 4.1 Yarrowia construct to E.coli.
15th – Thursday
- Miniprep of 4.1 Yarrowia construct from E. coli.
- Restriction verification of 4.1 Yarrowia construct.
- 1.6 construct transformation.
16th – Friday
- Cut and purify JMP1047 from gel.
27th – Tuesday
- Made 1L BG-11 liquid media.