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

For the construction of our Biosafety-System-Strain we deleted both Alanine-Racemases via homologous recombination with the pKO4 (a derivation from the pKO3 containing the coding sequence of the lacZ-alpha Fragment) to establish an D-alanine-auxotrophic mutant. For the deletion of the Alanine-Racemase we thereby used the following primers. The primers alr_Ec_d1 and alr_Ec_d2 will amplify an about 600 bp long homologous fragment upstream of the coding sequence of the Alanine-Racemase, while the primers alr_Ec_d3 and alr_Ec_d4 will perfom an about 600 bp long homologous fragment downstream of the Alanine-Racemase. This to deletion-sides can be ligated by Gibson-Assembly or alternative classically by gene SOEing (Gene splicing overlap Extension). The deletions-sides of the Alanine-Racemase were cloned into to vector by using the bold primer-overhang (see box below).
By following the protocol for the double-cross over using the heat-sensitive RepA and the sacB-Gene from the vector [http://arep.med.harvard.edu/labgc/pko3.html pKO3], the Alanine-Racemase could be successfully deleted.

Primer for the complete Deletion of the constitutive Alanin-Racemase (alr) and the catabolic Alanine-Racemase (dadX) in E. coli

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'

Primer: dadX_Ec_d1 (xx bp)

5'-TAGCTCACTCATTAGGCACCTGAAGTGTGGCGATGAAGT-3'

Primer:dadX_Ec_d2 (20 bp)

5'-GGGTCATCTCGTTTCCTTAG-3'

Primer: dadX_Ec_d3 (40 bp)

5'-CTAAGGAAACGAGATGACCCACTTGTTGTAAGCCGGATCG-3'

Primer: dadX_Ec_d4 (40 bp)

5'-GCTTTCTACGTGTTCCGCTTCGAAGCCAGCGCCAAATATG-3'

Primer for the control of the successful Deletion of the constitutive Alanin-Racemase (alr) and the catabolic Alanine-Racemase (dadX) in E. coli

Primer: alr_Ec_control1 (20 bp)

5'-GCTGGAGATGCCATCAGAAC-3'

Primer: alr_Ec_control2 (20 bp)

5'-CCGGCGAATATTGCTACGTG-3'

Primer: dadX_Ec_control1 (20 bp)

5'-GCTTTAATACGCCCGTTGAC-3'

Primer: dadX_Ec_control2 (20 bp)

5'-CTGGATCAACGCTTCTTTGG-3'

For the Deletion of the repressor araC we therefore used the same method as for the deletion auf die Alanin-Racemases in E. coli. The primers araC_d1 and araC_d2 will amplifiy analogous an about 600 bp long homologous fragment upstream of the coding sequence of the coding sequence for araC, while the primers araC_d3 and araC_d4 will perfom an about 600 bp long homologous fragment downstream of the araC gene. This two deletion-sides can also be ligated by Gibson-Assembly or gene SOEing (Gene splicin overlap Extension) and then cloned into to vector by using the bold primer-overhang (see box below). By following the protocol for the double-cross over using the heat-sensitive RepA and the sacB-gene from the vector pKO3, the Repressor araC could be successfully deleted.

Primer for the complete Deletion of the repressor araC for the arabinose promoter pBAD in E. coli

Primer: araC_d1 (40 bp)

5'-TAGCTCACTCATTAGGCACCCCGGCAGGAATATCGATCGC-3'

Primer: araC_d2 (40 bp)

5'-CTTCTCTGAATGGTGGGAGTGTCATAATTGGTAACGAATC-3'

Primer: araC_d3 (40 bp)

5'-GATTCGTTACCAATTATGACACTCCCACCATTCAGAGAAG-3'

Primer: araC_d4 (40 bp)

5'-GCTTTCTACGTGTTCCGCTTAACGCCAATCCCGACCACAG-3'

Results

References