Team:Chalmers-Gothenburg/Schedule

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Lab book compilation

June

9th
Preparation of 10x TE, 10x LiOAc, LiSorb, PEG/LiOAc and 200mM LiOAc + 1% SDS stocks

10th
Extraction of genomic DNA from Saccharomyces cerevisiae CEN PK 113 – 11C strain

17th
Inoculation of plasmids for all the constructs in Escherichia coli DH5α strain

18th
Purification of plasmids using plasmid miniprep kit

25th
Received all genes for Rec proteins in G-blocks, resuspended said G-blocks in TE buffer

29th
PCR to extract all fragments, for construct 4, 5, 6, 7 and 8 (Excl. Mam2 and Rec genes) from genomic DNA of S. cerevisiae.

30th
Inoculation of Schizosaccharomyces pombe (L972 h+ and Δ8 h- strains)
Extraction of genomic DNA from S. pombe L972 h+ strain
Gel electrophoresis on all PCR products
PCR purification of PCR products using PCR purification miniprep kit
Redone PCR of failed PCR products (TDH, PGK), also added Mam2 to PCR

July

1st
Gel electrophoresis on PCR (TDH, PGK and Mam2)
Nanodrop of purified PCR products
Preparation of S. pombe for cryo-storage
Linearization of plasmid (p0259 pXII-1-LEU) for construct 5
PCR purification of Mam2 using PCR purification miniprep kit
New PCR run on TDH and PGK

2nd
Gel electrophoresis of TDH and PGK
Gibson assembly on construct 5
PCR of TDH using both genomic DNA and PCR products (TDH) as template
Gel electrophoresis on PCR products (TDH)
Transformation of construct 5 into E. coli and incubation on plate for 16 hours

3rd
Gel electrophoresis on TDH using GelGreen
PCR on TDH
Gel electrophoresis on PCR products (TDH)
Took colonies from E. coli containing construct 5 for colony PCR and new plate
Gel electrophoresis on colony PCR

5th
Inoculation of colony 5 from colony PCR

6th
Purification of PCR products (TDH)
Linearization of plasmid (p0255-pX-2-URA)
Purification of plasmid containing construct 5 using plasmid miniprep kit
PCR on plasmid with construct 5
Gel electrophoresis on PCR products (construct 5)
Gibson assembly of RecJ, RecD2 and RecQ (Split on delivery in G-blocks)
Inoculation of S. cerevisiae CEN.PK2

7th
Gibson assembly of construct 4
Transformation of construct 4 into E. coli and incubation on plate for 16 hours
PCR on dCas9/Vp64, pCYC1m, and CGC1-Cas9 genes
Gel electrophoresis on PCR products (dCas9/Vp64, pCYC1m, and CGC1)
Gel electrophoresis for verification of construct 5
Purification of PCR products (construct 5)
PCR to amplify PprA, RecD2, RecJ, RecQ
Gel electrophoresis on PCR products (PprA, RecD2, RecJ, RecQ)

8th
PCR on pCYC1m using PCR products as template
Gel electrophoresis on PCR products (pCYC1m)
Purification of PCR products (CGC1) using miniprep
Took colonies from E. coli containing construct 4 for colony PCR and new plate
Restriction verification of plasmid with construct 5 using EcoRI and NotI
Linearization of plasmid with construct 5 using NotI
Preparation of –LEU YPD plates
Inoculation of plasmids (p0257 X-3-LEU, p0258 X-4-HIS, p0388 XI-1-LEU, p0391 XI-5-HIS) in E. coli
PCR to amplify PprA, RecD2, RecJ, RecQ
Gel on PCR products (PprA)

9th
Gel electrophoresis on Colony PCR (construct 4)
Inoculation of S. pombe
PCR on dCas9/Vp64
Gel electrophoresis on PCR products (dCas9/Vp64)
Colony PCR on construct 4 colonies 2, 3, 4, 10
Gel electrophoresis on colony PCR (construct 4) and PCR products (pCYC1m)
Gel electrophoresis on PCR products (pCYC1m) using GelGreen
Purification of plasmids (p0257, p0258, p0388, p0391) from E. coli
Gel electrophoresis on PCR products (PprA) using GelGreen
Gel electrophoresis on RecJ, RecQ, RecD2
Gel electrophoresis on PCR products (RecJ RecQ RecD2) using GelGreen

10th
PCR of dCas9/Vp64 using three different available templates
Gibson assembly of construct 1
Gel electrophoresis on Colony PCR (construct 4 centrifuged since yesterday)
Gel electrophoresis on PCR products (dCas9/Vp64) and construct 4
QuickYeast transformation of construct 5 into yeast
PCR on RecA and Ssb
Gel electrophoresis on PCR products (RecA and Ssb)

13th
Colony PCR of construct 4
Transformation of construct 1 into E.coli and incubation on plate for 16 hours
QuickYeast transformation of construct 5 into yeast
Linearization of plasmid (p0389) using AsisI
Gibson assembly of construct 6, 7 and 8

14th
Gel electrophoresis on Colony PCR (construct 4)
PCR on dCas9/Vp64 using PrimeStar polymerase (Previously Phusion HF)
Gel electrophoresis on dCas9/Vp64
Transformation of linearized plasmid (p0255) into E.coli for negative control of construct 1
QuickYeast transformation of construct 5 into yeast
Transformation of constructs 6, 7 and 8 into E .coli and incubation on plate for 16 hours
Preparation of –LEU YPD plates

15th
Linearization of plasmid (p0255 pX-2-URA) using AsiSI
PCR on pCYC1m and pFUS1
Gel purification of PCR products (dCas9/Vp64)
Gel electrophoresis on PCR products (pCYC1m and pFUS1) and linearized plasmid (p0255)
Linearization of plasmids using AsiSI and NotI
Transformation and Gel electrophoresis of negative control for construct 6, 7 and 8

16th
PCR on dCas9/Vp64
Gel purification of dCas9/Vp64
Linearization of plasmids (p0255, p0389) using NotI
Positive control of –LEU YPD plates

17th
Gel electrophoresis on PCR products (dCas9/Vp64) using GelGreen
Concentration of extracted products (dCas9/Vp64)
Preparation of –HIS plates
Verification of linearization of construct 5 plasmid using NotI

20th
Verification of linearization of construct 5 plasmid using NotI

21st
Restriction of p0259 using NotI for positive control
Inoculation of S. cerevisiae CEN.PK2

22nd
PCR purification of 2-pFUS1 and 3-pCYC1m
Transformation of construct 5 into yeast
Inoculation of plasmids (p0257, p0258, p0388, p0391) in E.coli
Plasmid linearization for constructs 1/4, 2, 3, 6, 7 and 8 using AsiSI

23rd
Purification of plasmids using miniprep
Inoculation of plasmids (p0255 and p0389) in E.coli
Plasmid linearization for constructs 2, 3, 7, 8 using AsiSI
Gel electrophoresis on linearized plasmids (constructs 2, 3, 7, 8) using GelGreen

24th
Concentration of linearized plasmids p0257, p0258, p0388, p0389
Gibson assembly of construct 7 and 8

27th
Purification and linearization of plasmids p0255, p0389
Transformation of constructs 7 and 8 into E .coli and incubation on plate for 16 hours

28th
Colony PCR of construct 7
Transformation of construct 8 into E. coli
Linearization of construct 5 (p0259) using NotI
Inoculation of construct 5 in yeast CEN.PK2

29th
Gel electrophoresis on colony PCR of construct 7
Transformation of construct 5 into yeast CEN.PK2
Gibson assembly of construct 4
Transformation of construct 4 into E. coli
Plating of construct 4 in E. coli

30th
Plating of construct 4 in E. coli
Colony PCR on construct 4 in p0257
Gel electrophoresis on construct 4
Suspension of BioBrick primers

31st
Verification of PUR parts
PCR on BioBricks
Gel electrophoresis on BioBricks
2nd Gel electrophoresis on BioBricks
PCR on backbone with primeSTAR

August

3rd
Inoculation of construct 4 in E. coli
Gel electrophoresis on RecJ using GelGreen
Gel electrophoresis on backbone and BioBricks
Transformation of construct 5 into yeast
PCR purification of plasmids

4th
Purification of plasmid containing construct 4 using plasmid miniprep kit
Gibson assembly of construct 1, 2, 7 and 8
Gel electrophoresis on products of Gibson assembly
Linearization of construct 4
Gel electrophoresis on restricted construct 4
Transformation of TEF in p425 into E. coli
Restriction prior yeast transform
PCR purification of BioBricks RecA, PprA, Ssb, SteMam

5th
2nd PCR of BioBricks RecA, PprA, Ssb, SteMam
Gel electrophoresis on 2nd PCR of BioBricks
Gel electrophoresis on construct 1, 2, 7 and 8
Transformation of construct 1, 2, 7 and 8 into E. coli
Inoculation of S. cerevisiae INFD-70 strain
Inoculation of E. coli

6th
Purification of plasmid (p425) using plasmid miniprep kit
PCR of construct 5 from gDNA
Gel electrophoresis on RecJ using GelGreen
Transformation of construct 1 into yeast
Linearization of construct 4
Preparation of 1M sorbitol

7th
Gel extraction of RecJ
Growing of S. pombe
Gel electrophoresis on construct 5 and RecJ
PCR on RecQ, RecD2, RecJ, RecA, PprA, Ssb
Genomic extraction of construct 5
Gel electrophoresis on RecQ, RecD2, RecJ, RecA, PprA, Ssb
PCR purification of RecQ, RecD2, RecJ, RecA, PprA, Ssb
Gel electrophoresis on RecJ

10th
Growing of S. pombe
Replated inoculation of construct 1, 2 and 8
2nd transformation of construct 4 into yeast
PCR on purified construct 5 (gDNA)
Gel electrophoresis on construct 5
Transformation of BioBrick vector into E.coli
Preparation of media for Safety Switch testing

11th
PCR on RecJ Purification of construct 1, 2 and 8 using miniprep
Nanodrop on construct 1, 2 and 8
Restriction of plasmids for construct 1, 2 and 8
Verification of construct 2 and 8

12th
Gel extraction of RecJ
Preparation of chloramphenicol plates
Testing Safety Switch
PCR using PrimeSTAR p0391 (8)

13th
OD measurements of Safety Switch and wildtype
Gel electrophoresis on PCR
PCR of BioBrick a-j04450
Test Safety Switch in YPG
Transformation of pSB1C3 into E .coli
Calculations on Safety Switch test

14th
Gel electrophoresis on PUR samples
Gibson assembly of construct 6, 7 and 8
PCR of Gibson assembly of construct 6, 7 and 8
Gel electrophoresis on construct 6, 7 and 8
Conduction of HPLC on samples from Safety Switch test
PCR purification of BioBrick vector
Restriction of BioBrick vector
Continued test Safety Switch in YPG

18th
Ligation of BioBricks
Transformation of BioBrick and construct 6, 7 and 8 into E.coli
Restriction of BioBrick vector

19th
Ligation of BioBricks
Gel electrophoresis on construct 6, 7 and 8
Re-plating and inoculation of construct 6 and 8
Restriction and ligation of BioBricks
Ligation of BioBrick TPI and CGC-CAS
Transformation of BioBrick TPI and CGC-CAS into E. coli

20th
Purification of construct 6 and 8
Transformation of BioBrick into E. coli

24th
Restriction of construct 6 and 8
Gel electrophoresis on construct 6 and 8
PCR of Safety Switch with RFP and BioBrick Safety Switch
Filtrating S. pombe

25th
Purification of plasmid containing BioBricks (RecA, Ste, Mam, SteMam)
Restriction of BioBricks inserts (RecA, Ste, Mam, SteMam)
Gel electrophoresis on restricted fragments for BioBricks (RecA, Ste, Mam, StêMam)
PCR of Safety Switch RFP
Restriction and ligation of BioBrick TEF and SUC
Gibson assembly of Safety Switch with RFP

26th
Gel electrophoresis of restriction fragments for BioBricks TEF and SUC
Transformation of BioBrick TEFSUC, SUC RFP, TEFSUC RFP into E. coli
Restriction and ligation of BioBricks
Sequencing of parts

27th
3rd transformation of construct 4 into yeast
Gibson assembly of construct 6, 7 and 8 without vector
PCR Gibson assembly of construct 6, 7 and 8 without vector
Transformation of BioBrick (26th) into E. coli
Gibson assembly of TEFSUC RFP and SUC RFP
Purification of BioBrick TEFSUC plasmid

28th
Restriction of construct 4 prior to transformation
2nd transformation of Safety Switch RFP to E. coli
Restriction of BioBrick TEFSUC 1-4
Gel electrophoresis on digested plasmid containing BioBrick TEFSUC 1-4
Restriction of plasmid containing BioBrick TEFSUC1
Gel of plasmid containing BioBrick TEFSUC1 digested with PstI, PstI + EcoRI

30th
Inoculation of construct 4 in CEN.PK2
Inoculation and re-plating of BioBrick TPI, SteMam, RecA, PprA and Ssb

31st
Purification of plasmids containing BioBrick TPI, SteMam, RecA, PprA and Ssb
Restriction of BioBrick TPI, SteMam, RecA, PprA and Ssb
Gel electrophoresis on BioBrick TPI, SteMam, RecA, PprA and Ssb
Re-plating and inoculation of Safety Switch with RFP
Inoculation and plating of construct 4 in CEN.PK2

September

1st
Genomic DNA extraction of construct 4 in CEN.PK2
PCR of construct 4 from CEN.PK2 gDNA
Restriction of TEFSUC insert
Restriction of BioBrick vector
Ligation of BioBrick TEFSUC2
Purification of Safety Switch with RFP using miniprep
Gel electrophoresis on Safety Switch with RFP
Restriction of Safety Switch with RFP

2nd
Gel electrophoresis of construct 4
1st test of construct 4
Transformation of Safety Switch SUC RFP and TEFSUC RFP into yeast CEN.PK2
Restriction of plasmid prior to RFP transformation
Transformation of BioBrick TEFSUC into E. coli

3rd
Concentration of pheromones
Overnight cultivation of CEN.PK2 with m- and p-factor pheromones from S. pombe
Inoculation of construct 4 in E. coli
OD measurements for Safety Switch TPK2

4th
2nd test of Safety Switch TPK2 and WT
Restriction of BioBrick vector and TEFSUC
Ligation of BioBrick TEFSUC with BioBrick vector

7th
Preculture drop test Safety Switch TPK2
Re-plating Safety Switch RFP

8th
Cultivation of cells for spectrometry
PCR of BioBrick TEFSUC and Vector J04450
Preparation pf chloramphenicol plates
Gel electrophoresis on BioBrick TEFSUC and vector J04450
Nanodrop of BioBrick TEFSUC and BioBrick vector
Restriction of BioBrick TEFSUC and BioBrick vector

9th
2nd test of construct 4
Testing BioBricks
Genomic extraction Safety Switch TEF RFP and SUC RFP
Colony PCR Safety Switch TEFRFP and SUC RFP
Gel of Safety Switch RFP and SUC RFP
Colony PCR of BioBrick RFP and SUC RFP
Restriction and ligation of BioBrick TEFSUC
Transformation of BioBrick TEFSUC into E. coli

10th
Restriction of BioBrick and vector
Purification of BioBrick and vector

11th
Concentration of p-factor pheromone
1st droptest on Safety Switch TPK
2nd droptest on Safety Switch TPK
Inoculation of WT, CEN.PK2, Safety Switch TPK2-Col 1, TEFSUC RFP, SUC RFP and RFP
Re-plating and inoculation of E. coli with BioBrick TEFSUC

12th
3rd test of construct 4
Replating of E. coli with BioBrick vector p

14th
Miniprep of E. coli with BioBrick vector from plate
Restriction of product from miniprep (BioBrick vector)
Restriction of BioBrick parts
Gel electrophoresis on cut vector
Ligation of BioBricks in BioBrick vector
Transformation of BioBricks into E. coli and let to grow on plate
Preparation of chloramphenicol plates

16th
Inoculation of E.coli colonies containing BioBricks

17th
Purification of BioBrick using plasmid miniprep kit
Restriction of BioBricks
Gel electrophoresis on BioBricks

128h
Drying of successful BioBricks for submission to iGEM