OUR LONG SUMMER!
- Installation and cleaning of the academic laboratory
- verification of equipments (calibration…)
- start of interlab’s experiments
- design of our pBullet plasmid, capable of conjugation and recombination of a Crispr/Cas9 system, targeting RFP and AmpR gene, with a biosafety component (Lambda-Pir dependent)
- Complete modification of our plasmid’s design
- Culture and Minipreps of the pCas plasmid
- Enzyme digestion of Cas9 gene ( promoter and terminator too)
- Phenol/Chloroform purification of the Cas9 cassette
- Preparation of electrocompetent cells (S17-1) to receive our plasmid
- Order (at IDT) for our synthetic sgRNA part (targeting RFP and AmpR gene)
- Culture and phenol/Chlo purification of the plasmid pK18mob (our mobilizable plasmid, which will receive the Cas9 and sgRNA)
- Minipreps of the pK18mob plasmid
- PCR to amplify the Cas9 gene (with tracer part) of pCAS plasmid
- Single/Double enzymatic digestion of pK18mob plasmid to check
- Minipreps, Proteinase K and Phenol/Chloroform purification after a PCR of Cas9
- Double enzymatic digestion of the Cas9 cassette
- Ligation of the Cas9 cassette into the pK18mob plasmid + verification of the result with enzymatic digestion and PCR
- Minipreps of the new plasmid (pK18mob+Cas9 cassette)
- Transformation of the new plasmid into the pUC19 strain
- PCR and purification of the Cas9 cassette (with the tracer part of the Cas9)
- Receipt of the IDT order (our synthetic sgRNA targeting RFP and AmpR)
- Stability test of our receiver plasmid (low copy plasmid pSB4K5_BBa_J04450)
- Enzymatic digestion (linearization) of our biosafety plasmid (pSW23T carrying R6K Ori and Lambda-Pir dependent)
- Enzymatic digestion of the Cas9 cassette (tracer version) and ligation into pK18mob plasmid
- Verification of the sgRNA (received from IDT) by Miniprep and double enzymatic digestion
- Transformation of the sgRNA (with and without NheI site) (note: 3 of the 6 orders received from IDT was empty !)
- Transformation of the new pK18mob into chemically competent cells
- Glycerol stock and Minipreps of the sgRNA
- Glycerol stock and Minipreps of the pK18mob with Cas9 cassette (tracer version)
- Verification by enzymatic digestion of the plasmid insertion
- Double enzymatic digestion of the sgRNA targeting RFP and the new pK18mob (with Cas9 cassette)
- Ligation of the sgRNA targeting RFP with the new pK18mob (with Cas9 cassette)
- First test to check the conjugation protocols
- Minipreps and gel verification of the sgRNA targeting RFP and the new pK18mob (with Cas9 cassette)
- Sequencing of the pK18mob + Cas9 cassette and sgRNA (with and without NheI)
- Transformation of the new plasmid (pK18mob + Cas9 cassette + sgRNA targeting RFP)
- Verification of all our strain with antibiograms
- PCR modification, minipreps and gel verification of the plasmid pSW23T to modify the MCS
- Sequencing of the pK18mob + Cas9 cassette (tracer version)
- Concentration verification (Nanodrop) of our killer plasmid (targeting RFP gene)
- Transformation into S17-1 of our killer plasmid (targeting RFP gene)
- Verification of efficiency (PCR diagnostic, enzymatic digestion, gel) of the BaeI enzyme (for our new biobrick system)
- PCR diagnostic of our killer plasmid (two version of the Cas9 with and without tracer)
- New clonage approach test for the sgRNA (NheI version) with pGem T-easy
- Verification test of the efficiency of the Cas9 and sgRNA in pK18mob plasmid + stability test of our killer plasmid with our reporter plasmid.
- Electroporation and PCR of our killer plasmid in S17-1 Lambda-Pir
- PCR diagnostic of the pGem T easy
- Receipt of order from IDT of the sgRNA targeting AmpR gene.
- Enzymatic digestion and verification of the sgRNA targeting AmpR gene.
- Ligation and electroporation of our plasmid pK18mob with Cas9 cassette and the new sgRNA targeting AmpR gene.
- Interruption of all manipulation concerning both sgRNA (with and without NheI site) and the Cas9_tracer_version. (No DNA in the IDT order)
- PCR verification after transformation of our new plasmid targeting AmpR gene + glycerol stock
- Minipreps and enzymatic digestion to check the presence of the sgRNA
- Verification of Crispr/Cas9 efficiency of our killer plasmid
- Enzymatic digestion, ligation, electroporation and verification of our biosafety plasmid (pSW23T targeting RFP and AmpR gene)
- Transformation + verification of our differents plasmids in E. coli strains.
- Sequencing of our killer plasmid targeting AmpR gene.
- First test of conjugation in a liquid phase
- Cleaning of the academic laboratory
- Gibson + PCR amplification of pUC19
- Verification of the IDT order (our new biobrick)
- Transformation and PCR verification of the insertion of the Cas9+sgRNA cassette in the new pSW23T.
- Verification of the new IDT order (sgRNA with GFP reporter gene) : No DNA.
- Second verification of the efficiency of our Crispr/cas9 system
- Test of selection of our donor and receiver strain over NAC.
- Conjugation test 2 in liquid phase
- Conjugation test in plate with IPTG/X-Gal system
- Gel control of the last conjugation
- Conjugation test of our donor strain (S17-1 with our killer plasmid targeting AmpR gene) and a E.coli strain carrying TEM1 gene in his chromosome.
- Enzymatic digestion and verification of a new reporter plasmid (with GFP AmpR and pSC101)
- Conjugation test with differents donor and receiver strains
- Verification of conjugation efficiency between donor strain and receiver strain, with IPTG/X-Gal system
- preparation of strains for fluorescent microscopy
- Fluorescent microscopy assay
- Conjugation “in plate” test with differents donor and receiver strains
- Quantitative conjugation “in plate” test with differents donor and receiver strains
- Conjugation “cross” test between our donor and receiver strains.
- Wiki writting
- Wiki writting and preparation of the poster
- Cleaning of the L2 laboratory