Molecular Evolution Platform used for Taq polymerase
Technology based on site directed mutagenesis of the gene for Taq polymerase and then the selection of the mutated enzyme by its resistance to pressure imposed
The pressure varies according to objective (robsutesse, speed ...)
Principle of technology
| 1. Cloning of
the Taq polymerase 2a. random mutagenesis 2b. Cloning of mutants in E. coli 2c. Production of the mutated Taq polymerase 3a. Resuspension of E. Coli in soluble phase on a hydrophobic phase 3b. emulsion 4a. PCR under certain pressure conditions: amplification of mutant clones selected 4b. Visualization of the amplification obtained |
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Principle of selection
 |
| 1 - Each bacterium contains a plasmid encoding a mutant
wild type Taq and
is resuspended in a
medium containing the primers
amplifying the mutated
Taq 2 - Each bacterium is isolated by emulsion in a bubble containing medium-soluble 3 - PCR in the presence of a pressure condition (destruction of the bacteria by heat) 4 - Amplification of the amplicons encoding a Taq resistant pressure conditions 5 - Merging all the drops to get the starting soluble medium 6 - Sequencing of mutant candidates for a second round of selection |
Pressure conditions
Kapa 2G Robust
-
Addition of NaCl in the soluble medium
- Addition of urea in the soluble medium
- Addition of SDS in the soluble medium
- Addition of ethanol in the soluble medium
Kapa
2G Fast
- Stop of elongation after 3 secondes
Pour
la Kapa
Plant PCR kit
- Addition of polyphenols in the soluble medium
Pour
la Kapa
SYBR Fast
kit
- Addition of SYBR Green in the soluble medium