SIG.b/LMGM

Internal development (2021):

LMGM bought the ASKA collection of Escherichia coli strains (individual clones in microplate format). Each of the 4123 predicted ORFs of E. coli W3110 has been amplified by PCR, inserted into a plasmid under control of an –inducible promoter and established in E. coli strain AG1 by transformation.

To carry out multicopy suppressor selection experiments, SIG.b made a library of plasmids representative of this bank. The library has been validated in 2 ways:

– Complementation assay on 5 auxotrophic deletion mutants: transformation with the ASKA sub libraries and sequencing of the plasmid content of the resulting colonies.

– Whole Direct Sequencing (Nanopore, outsourced service).

Collaborative project (2023, unpublished):

Description:

In 2023, we started a collaboration with C. Albenne from LMGM aimed at understanding the function of protein X, an outer membrane protein. Deletion of X causes loss of fitness and vancomycin susceptibility.

Service Objectives:

Our goal was to identify multi-copy suppressors by determining which of the AKSA plasmids improved vancomycin resistance when introduced by transformation in an X gene deletion mutant (in the presence of the inducer).

Of 910 plasmids analysed by spot test, PCR, gel electrophoresis and sequencing, a list of 77 candidates was provided to the collaborative team.

Funding:

Costs (consumables and sequencing fee) were covered by the collaborating team.