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 X, 2014
TEX
July X, 2014
TEX

RNAi

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YFP

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CONJUGATION

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OTHER

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