Safety Strain

Overview

We produced a auxotrophic safety strain which was the basic for our biosafety system. The strain consists Δdadx and Δalr knockouts which prevents the expression of alanine racemase. Alanine racemase is an isomerase that catalyzes the chemical reaction from L-alanine to D-alanine. D-alanine appears in peptidoglycan which is an important component of the bacterial cell wall. So the safety strain depends on the supplementation of alanine racemase on the plasmid which causes a good plasmid stability. If the bacteria cast the plasmid it will die because of the fact that L-alanine couldn’t be isomerized into D-alanine anymore and the cell wall wouldn’t be assemble in the correct way.

Theory

Genetic Approach

Primer

Primer: alr_Ec_d1 (39 bp)

5'-TAGCTCACTCATTAGGCACCCAGCTCGATGACGAAGACT-3'

Primer: alr_Ec_d2 (20 bp)

5'-GCCGCTTGCATTTGTGTTCC-3'

Primer: alr_Ec_d3 (40 bp)

5'-GGAACACAAATGCAAGCGGCTTGATTGTCTGTGCCGGATG-3'

Primer: alr_Ec_d4 (40 bp)

5'-GCTTTCTACGTGTTCCGCTTCCGGGAAGTAGCGTTTCAGG-3'

Results

References