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Plasmid stability test
Immediately before indcution, it is advisable to test the culture to determine the fraction of cells that still carry the target plasmid. This involves plating of cells on four different plates.
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Plate |
cells that grow on these plate |
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LB plate |
all viable cells |
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LB plate + antibiotic |
cells that still carry the plasmid |
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LB plate + IPTG (1 mM) |
cells that have lost the plasmid or mutants that have lost the ability to express the target gene |
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LB plate + antibiotic + IPTG (1 mM) |
only mutants that retain the plasmid but have lost the ability to express the target gene |
Remarks
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In the presence of IPTG, cells carrying a protein production plasmid do not grow because have dedicated all their resources to the production of the recombinant protein instead of cell maintainance.
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In the presence of the pLysS vector, IPTG also prevents colony formation except with certain vector (such as pET-3 and some vectors carrying the T7lac promoter). In the presence of pLysE, IPTG usually does not prevent colony formation unless the target protein is toxic.
In a typical culture useful for producing target protein, almost all cells will form colonies both on the LB plate and on the LB plate + antibiotic; less than 2% of the cells will form a colony on the LB plate + IPTG; and less than 0.01% will form a colony on the LB plate + antibiotic + IPTG.
With unstable target plasmids, the fraction of cells that have lost the plasmid will be reflected by an increase in colonies on the LB plate + IPTG and a decrease on ther LB plate + antibiotic.
Protocol
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1. |
Immediately before induction with IPTG (at OD600 is approx. 0.6), take a 100-ml aliquot of the cell culture. |
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2. |
Make a serial dilution of the cell suspension, including a 105 and 106 dilution. |
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3. |
Plate cells at a dilution of 105 on the LB plate + IPTG and on the LB plate + IPTG + antibiotic. |
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Plate cells at a dilution of 106 on the LB plate and on the LB plate + antibiotic. |
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4. |
Incubate the plates overnight a 37°C. |
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5. |
Count the number of colonies on each plate. |
Reference: pET System Manual, 8th ed., 1999 (Novagen).
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