C43(DE3)pLysS Competent Cells

1. Competent cells should be used immediately after thawing and should not be left in ice for too long.
2. Do not aspirate competent cells with a pipette，just flick your finger to mix well.
3. When inducing protein expression，the IPTG concentration is optional (0.1-2 mM). This product is a competent product made of Escherichia coli BL21Star (DE3). Both strains，OverExpress C43 (DE3) and OverExpress C41 (DE3)，originated from BL21 (DE3)，have the advantage of efficient expression of toxic or hydrophobic proteins. The difference between OverExpressC41 (DE3) and BL21 (DE3) is that its genome contains at least one unknown mutation，which gives it the ability to efficiently express toxic proteins，and this mutation site is involved in the cell death pathway when E. coli expresses toxic proteins. OverExpressC43(DE3) is derived from OverExpress C41(DE3) and was obtained by screening for strains resistant to another different virulence protein of OverExpress C41(DE3). The C43 (DE3) strain has a stronger ability to express toxic and hydrophobic proteins than C41 (DE3). The chloramphenicol-resistant pLysS plasmid was introduced into C43(DE3) cells，that is，C43(DE3) pLysS，pLysS expressed T7 lysozyme，which could bind to T7 RNA polymerase and inhibit its transcriptional activity，thereby reducing the background expression level of the target gene，but did not interfere with the expression induced by IPTG，which was very suitable for the prokaryotic expression of toxic proteins. The chromosome of this strain integrates the lambda phage DE3 region (the DE3 region contains T7 bacteriophage RNA polymerase)，which can simultaneously express T7 RNA polymerase and E. coli RNA polymerase，and can be used for protein expression of pET series，pGEX，pMAL and other plasmids. C43(DE3)pLysS competent cells were fabricated by a special process，and the transformation efficiency of the pUC19 plasmid was > 1×108cfu/μg DNA. Genotype: F–ompThsdSB(rB-mB-) gal dcm (DE3)pLysSCamR Procedure: 1. Remove the competent cells from the -80℃ freezer，put them in ice for 5 min，add the plasmid of interest or ligation product，flick to mix evenly，and incubate in ice for 30 min; 2. Put it in a 42°C water bath for 90s，immediately insert it into ice and let it stand for 2-3min; 3. Add 900 μL of LB liquid medium and incubate at 220 rpm at 37℃ for 60 min; 4. Centrifuge at 5000 rpm for one minute to collect bacteria，leave about 100 μl of supernatant and gently pipette the resuspended bacterial block and apply it to a 2YT or LB plate containing the corresponding antibiotic，and place in an incubator at 37°C overnight.