Team:Chalmers Gothenburg/Experiments/Lab journal

Chalmers Gothenburg iGEM 2016

NOTEBOOK
Lab Journal

June

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.
  • PCR's.
  • 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.

July

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.
  • PCR.

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
    • BBa_K823003
    • BBa_E0840
    • pKD3
    • p416TEF
  • 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
    • 1.4acsUp+spr
    • 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.

August

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.

September

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.