PlasmidstABIlitytestImmediatelybeforeindcution,itisadvisabletotesttheculturetodeterminethefractionofcellsthatstillcarrythetargetplasmid.Thisinvolvesplatingofcellsonfourdifferentplates.
RemarksPlate cellsthatgrowontheseplate LBplate allviablecells LBplate+antibiotic cellsthatstillcarrytheplasmid LBplate+IPTG(1mM) cellsthathavelosttheplasmidormutantsthathavelosttheabilitytoexpressthetargetgene LBplate+antibiotic+IPTG(1mM) onlymutantsthatretaintheplasmidbuthavelosttheabilitytoexpressthetargetgene
- InthepresenceofIPTG,cellscarryingaproteinproductionplasmiddonotgrowbecausehavededicatedalltheirresourcestotheproductionoftherecombinantproteininsteadofcellmaintainance.
- InthepresenceofthepLysSvector,IPTGalsopreventscolonyformationexceptwithcertainvector(suchaspET-3andsomevectorscarryingtheT7lacpromoter).InthepresenceofpLysE,IPTGusuallydoesnotpreventcolonyformationunlessthetargetproteinistoxic.
Inatypicalcultureusefulforproducingtargetprotein,almostallcellswillformcoloniesbothontheLBplateandontheLBplate+antibiotic;lessthan2%ofthecellswillformacolonyontheLBplate+IPTG;andlessthan0.01%willformacolonyontheLBplate+antibiotic+IPTG.
Withunstabletargetplasmids,thefractionofcellsthathavelosttheplasmidwillbereflectedbyanincreaseincoloniesontheLBplate+IPTGandadecreaseontherLBplate+antibiotic.
Protocol
| 1. | ImmediatelybeforeinductionwithIPTG(atOD600isapprox.0.6),takea100-mlaliquotofthecellculture. |
| 2. | MakeaserialdilutionofthecellsUSPension,includinga105and106dilution. |
| 3. | Platecellsatadilutionof105ontheLBplate+IPTGandontheLBplate+IPTG+antibiotic. |
| Platecellsatadilutionof106ontheLBplateandontheLBplate+antibiotic. | |
| 4. | Incubatetheplatesovernighta37°C. |
| 5. | Countthenumberofcoloniesoneachplate. |
