CRISPRi

June 6, 2014
Digested pSB1C3 backbone with EcoRI and PstI and gel purified
June 9, 2014
Received synthesized sgRNA 1 and sgRNA 2 constructs from BioBasic and transformed the DNA into E. coli DH5α
June 11, 2014
Inoculated sgRNA 1 and 2 colonies from the transformation into over night liquid culture for frozen stocks and miniprep
June 18, 2014
Digested sgRNA 1 and 2 constructs with EcoRI and PstI, then gel purified the inserts for cloning into pSB1A3 Also digested sgRNA 1 with EcoRI and SpeI, and sgRNA 2 with XbaI and PstI for modified 3A assembly into pSB1A3
June 19, 2014
Digested pSB1A3 linearized backbone with EcoRI and PstI, then column purified.
Ligated pSB1A3 with the sgRNA 1 and 2 (E/P) inserts individually and with both sgRNA 1 (E/S) and sgRNA 2 (X/P) together
Ligated pSB1C3 backbone with sgRNA 1 (E/P) and sgRNA 2 (E/P) individually for BioBricks
June 20, 2014
Transformed all ligations into E. coli DH5α
June 21, 2014
Streak purified 4 colonies from each ligation
June 22, 2014
Inoculated 4 colonies of each ligation into overnight liquid culture for miniprep (patched colonies onto agar plates before inoculating
June 23, 2014
Miniprepped and did a diagnostic digest to see which clones contained the appropriate inserts: -sgRNA 1 in pSB1A3: 1 successful
-sgRNA 2 in pSB1A3: 3 successful
-sgRNA 1 and sgRNA 2 in pSB1A3: 0 successful
-sgRNA 1 in pSB1C3: 1 successful
-sgRNA 2 in pSB1C3: 2 successful
June 24, 2014
Streak purified 4 more colonies from the sgRNA 1 and sgRNA 2 in pSB1A3 ligation to screen for successful clones
June 25, 2014
Inoculated the 4 clones of sgRNA 1 and sgRNA 2 in pSB1A3 for miniprep (patched colonies onto agar plates before inoculating)
June 26, 2014
Miniprepped and did a test digest to screen for clones with the correct insert size: 0 successful
Decided to wait for the sgRNA 1 in pSB1A3 to be sequence confirmed and then clone in sgRNA 2 instead of doing modified 3A assembly
July 3, 2014
Inoculated successful clones from patches into over night liquid culture to miniprep and send for sequencing
July 4, 2014
Miniprepped and sent clones for sequencing
July 9, 2014
Received synthesized dCas9 cassette from BioBasic and transformed the DNA into E. coli DH5α
July 10, 2014
Digested the dCas9 construct with EcoRI and PstI then gel purified the insert
Digested more pSB1A3 and pSB1C3 with EcoRI and PstI and gel purified the backbone.
Ligated the dCas9 cassette (E/P) into pSB1A3 (E/P) and pSB1C3 (E/P)
July 11, 2014
Transformed all ligations into E. coli DH5α
July 12, 2014
Streak purified 8 colonies from each ligation
July 13, 2014
Inoculated the colonies into over night liquid cultures for miniprep (patched colonies onto agar plates before inoculating)
July 14, 2014
Miniprepped and did a diagnostic digest to see which clones contained the appropriate inserts:
- dCas9 cassette in pSB1A3: 0 successful
- dCas9 cassette in pSB1C3 : 2 successful
Ran digests of sgRNA for gel extraction
July 15, 2014
Streak purified 8 colonies from dCas9 pSB1A3
Gel extracted sgRNA
Restriction digest and gel extraction of dsRed-ermM-oriV, ermM-oriV, pSB1C3, pSB1A3
July 16, 2014
Inoculated and patched 8 colonies of dCas9 pSB1A3
Miniprepped sequence-confirmed sgRNA strains, pSB1C3, pSB1A3, ermM-oriV, dsRed-ermM-oriV
Digested, column purified, and ligated
July 17, 2014
Miniprep and restriction digest of 8 colonies of dCas9 pSB1A3: 0 successful
Transformed ligations of sgRNA, pSB1C3, pSB1A3, and ermM-oriV
July 18, 2014
Streak purified 4 colonies for each ligation
July 19, 2014
Miniprepped and digested:
sgRNA, ermM-oriV, sgRNA-ermM-oriV, dCas9-ermM-oriV
sgRNA1+sgRNA2+pSB1A3: 2 successful
sgRNA1+pSB1C3: 4 successful
ermM-oriV-pSB1A3: 1 successful
sgRNA1-ermM-oriV-pSB1A3: 0 successful
sgRNA2-ermM-oriV-pSB1A3: 1 successful
dCas9-ermM-oriV-pSB1A3: 2 successful
July 22, 2014
ermM-oriV PCR
July 24, 2014
ermM-oriV restriction digest and ligation in pSB1A3
Miniprep and digest: spc construct-pUC57, dCas9 cassette
dsRed-ermM-oriV in pUC57 digest and gel extraction
July 25, 2014
dsRed-ermM-oriV digest and transformation into DC10B
Transformation: ermM-oriV-pSB1A3 4 colonies
July 27, 2014
Miniprep and digest of:
ermM-oriV-pSB1A3: 3 successful
dsRed-ermM-oriV-pUC57-DC10B: 3 successful
July 28, 2014
Gel extracted dCas9-ermM-oriV
July 29, 2014
Ligation of dCas9-ermM-oriV with sgRNA
July 30, 2014
Restriction digest, column purification and ligation of dCas9-ermM-oriV and sgRNA
July 31, 2014
Transformed and inoculated dCas9-ermM-oriV to DH5α and pSB1A3
August 1, 2014
Miniprepped and picked colonies from transformation, then grown in LB + Amp
August 2, 2014
Miniprepped and digested:
dCas9-ermM-oriV+sgRNA1: 1 successful
dCas9-ermM-oriV+sgRNA2: 1 successful
dCas9-ermM-oriV+sgRNA1+sgRNA2: 2 successful
dCas9-ermM-oriV+pSB1A3: 1 successful
August 5, 2014
PCR of ermM-oriV
August 6, 2014
PCR ermM and dsRed: 0 successful
Ligation and digest for ermM-oriV, sgRNA and pSB1A3
ermM-oriV column purified
August 8, 2014
Transformation of ermM-oriV+pSB1A3 into DH5α
Many colonies, of which 6 were picked
PCR ermM and dsRed
August 9, 2014
Miniprep and digest of ermM-oriV+pSB1A3: 0 successful
August 11, 2014
ermM and dsRed column purified: 0 successful
August 12, 2014
Transformed:
DsRed-ermM-oriV into DH5α
DsRed CDS into pSB1C3
ermM CDS into pSB1C3
Colony PCR of:
ermM-oriV+pSB1A3
dCas9-ermM-oriV+pSB1A3
August 13, 2014
Miniprepped, made stocks and picked colonies:
sgRNA1-dCas9-ermM-oriV
sgRNA2-dCas9-ermM-oriV
sgRNA1-sgRNA2-dCas9-ermM-oriV
Inoculated:
YFP construct
aadD construct-pUC57
spc construct-pUC57
dsRed-ermM-oriV-pUC57 in DH5α
ermM-oriV-pSB1A3
dCas9-ermM-oriV-pSB1A3
August 14, 2014
Restriction digest of inoculated samples: 4 successful
August 19, 2014
Digested dsRed-ermM-oriV
September 4, 2014
Transformed J23101 and GFP into DH5α and plated on kanamycin plates
Streak purified DsRed-ermM-oriV and sgRNA from stock
September 5, 2014
Testing erythromycin resistance in E. coli
DsRed-ermM-oriV_{S. aureus} in pUC57, DC10B
sgRNA in pUC57, DH5α (negative control)
negative control had growth, so erythromycin stock was remade
PCR of dsRed
September 6, 2014
Inoculated and miniprepped J23101, GFP
September 8, 2014
PCR of xylose promoter: no products
oriV – PCR, column purification, and digest with PstI and EcoRI
September 9, 2014
Ligation of oriV
Colony PCR for ermM-oriV+pSB1A3 and dCas9-ermM-oriV+pSB1A3: no amplification seen
Gel extraction of DsRed-ermM-oriV
September 10, 2014
Colony PCR testing KOD and Taq
PCR of xylose promoter
September 11, 2014
Gel extraction of xylose promoter
Inoculated oriV to LB with chloramphenicol
September 12, 2014
Digested xylose promoter with EcoRI and SpeI
Digested oriV pSK41 with EcoRI and PstI
Restriction digest and ligation of ermM-oriV with pSB1A3: 0 successful
September 13, 2014
Heat shock transformation of ermM-oriV + pSB1A3 into DH5α and gel extracted
September 14, 2014
Restriction digest, gel extraction, and ligation of ermM-oriV-pSB1A3 in DH5α
Inoculation and 10 colonies picked
PCR of dsRed-oriV-ermM
September 15, 2014
Gel extraction of PCR
Miniprep of 10 colonies
PCR, diagnostic digest of miniprepped ermM-oriV-pSB1A3: 2 successful
September 16, 2014
Digest of oriV: 0 successful
Streak purified:
pSB1C3+RFP
J23101-GFP
sgRNA
dsRed-ermM-oriV
Inoculated and patched 12 oriV-pSB1C3 colonies
September 17, 2014
Xylose promoter-GFP miniprep and digest: 0 successful
ermM-oriV-pSB1A3 digest: 0 successful
Restriction digest of sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-dCas9-ermM-oriV cut with EcoRI and PstI: 1 successful
September 18, 2014
Miniprep and restriction digest of oriV: 7 successful
September 19, 2014
Xylose promoter and dsRed PCR: successful
Miniprep of ermM-oriV and sgRNA1-dCas9-ermM-oriV
September 22, 2014
Gel extraction and PCR of xylose promoter, dsRed, ermM
Restriction digest of ermM-oriV
PCR of pSB1C3 and dsRed-ermM-oriV
September 23, 2014
Miniprep of xylose promoter, oriV
Restriction digest of xylose promoter: 0 successful
Gel extraction of pSB1C3 and dsRed-ermM-oriV
Restriction digest of pSB1C3 shuttle vector, ermM-oriV shuttle vector, sarA promoter, xylose promoter, dsRed cassette, ermM cassette
Heat inactivated sarA promoter
Column purified all the digests
September 24, 2014
GFP miniprep and digest with EcoRI-HF and XBaI
Ligations of digests
Inoculation of J23101-GFP, dsRed-ermM-oriV, sgRNA to check fluorescence
Transformation into DH5α: shuttle vector, xylose promoter-pSB1C3, dsRed, ermM, sarA promoter, xylose promoter, shuttle vector control, pSB1C3 control, GFP control
September 27, 2014
PCR of ermM-oriV and pSB1A3
Restriction digest:
xylose promoter-GFP with 5 successful
sarA promoter-GFP with 5 successful
shuttle vector with RFP with 1 successful
xylose promoter with 1 successful
GFP (negative control)
Inoculated and patched ermM-oriV-pSB1A3
Inoculated oriV, xylose promoter-GFP, sarA promoter-GFP, shuttle vector-RFP, sgRNA1, sgRNA2
September 27, 2014
PCR of pSB3K3, ermM-oriV with prefix/suffix, ermM-oriV with BamHI/BglII, pSB1A3 with BamHI/BglII
Miniprepped oriV, xylose promoter-GFP, sarA promoter-GFP, shuttle vector-RFP, xylose promoter
October 1, 2014
Inoculated xylose promoter-GFP, sarA promoter-GFP, J23101-GFP, DH5α
Miniprep of dsRed-ermM-oriV and RFP
October 2, 2014
Diluted cultures 1/100 and incubated for 3 hours and read with plate reader for fluorescence and again after half an hour for xylose promter-GFP, xylose promoter-GFP with xylose, sarA promoter-GFP, J23101-GFP, DH5α
Fluorescence seen in J23101-GFP, sarA promoter and in xylose promoter-GFP with xylose
October 3, 2014
Digest of dsRed-ermM-oriV
October 5, 2014
Streak purified DH5α, J23101-GFP, GFP, xylose promoter-GFP, sarA promoter-GFP
October 9, 2014
Inoculated sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-ermM-oriV, dCas9-ermM-oriV
Inoculated J23101-GFP, GFP, DH5α, xylose promoter-GFP, sarA promoter-GFP, dsRed-ermM-oriV_{S.epidermidis}, dsRed-ermM-oriVDC10B, S.epidermidis, YFP cassette, YFP-F, hfq cassette, oriV, RFP, xylose promoter, sarA promoter
Digest of dCas9-ermM-oriV, sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-dCas9-ermM-oriV
October 10, 2014
Gel extraction of dCas9-ermM-oriV, sgRNA1-dCas9-ermM-oriV, sgRNA1-sgRNA2-dCas9-ermM-oriV
Ligation of dCas9-ermM-oriV in pSB1A3
Transformation of dCas9-ermM-oriV-pSB1A3 into DH5α and DC10B
Measure absorbance spectra for dsRed-ermM-oriV
October 13, 2014
Ligation of dCas9-ermM-oriV and pSB1A3
October 14, 2014
Digest for CRISPR assay
co-transformation successful
Promoter characterization:
3 colonies each :xylose promoter-GFP, sarA promoter-GFP, J23101-GFP, GFP, DH5α, xylose promoter-GFP with xylose
Inoculated from overnight cultures into 5ml LB in 1/100 dilution with antibiotics
Incubated for 3 hours and read absorbance and fluorescence on plate reader
Xylose promoter unsuccessful – use fresh xylose next time
Reinoculated cultures for next day
October 15, 2014
CRISPR assay
Inoculated overnight cultures to fresh LB + antibiotic + xylose in triplicate at 1/100 dilution
Grew until 0.5-0.6 OD and read fluorescence with plate reader unsuccessful

RNAi

June 10, 2014
Received pJB38 integrative plasmid, pKAN, pSPC, pYFP-F, pYFP-R, pRFP-F, and pRFP-R from Dr. Bose, Dr. Bayles, and Dr. Endres from the University of Nebraska, Nebraska, U.S.A in E. coli DH5α. Inoculated colonies into over night liquid cultures for frozen stocks and miniprep
June 16, 2014
Received synthesized YFP construct (1st half of integration insert) from BioBasic and transformed the DNA into E. coli DH5α
June 18, 2014
Inoculated 4 colonies of the YFP construct from the transformation into over night liquid culture for frozen stocks and miniprep
June 19, 2014
Minipreped the 4 cultures and did a diagnostic digest before making frozen stocks
June 20, 2014
Received the synthesized 2nd half of the integration insert containing the aadD gene from BioBasic. Transformed DNA into E. coli DH5α
June 21, 2014
Streak purified 4 colonies of aadD containing construct
June 22, 2014
Inoculated the 4 colonies of aadD containing construct into over night liquid cultures for miniprep and frozen stocks
June 23, 2014
Minipreped the 4 cultures and did a diagnostic digest before making frozen stocks Digested the following for modified 3A assembly:
- YFP construct with EcoRI and SpeI
- aadD containing construct with XbaI and SalI
- pJB38 backbone with EcoRI and SalI
Gel purified the inserts and ligated all the three parts together
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YFP

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CONJUGATION

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OTHER

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