Core facility
Presentation
The Bacterial Genetics Engineering Department, certified in 2025 by the GIS IBiSA, operates from two sites in Toulouse (LMGM-CBI and IPBS). It is a genome engineering resource center with two missions:
1/ In order to specifically respond to the problems of research teams, the SIG.b platform designs and implements bacterial molecular genetics approaches (conventional genetic engineering tools and tools derived from CRISPR systems) adapted to various bacterial strains, whether they are model strains or not, pathogenic or clinical strains.
We work in collaborative projects with teams from public or private laboratories, French or foreign.
Depending on the strain studied, we carry out an in-depth bibliographic analysis, making it possible to propose different genome editing or screening strategies.
If it is a strain that is still poorly characterized, we develop the culture conditions, growth monitoring and enumeration, develop the appropriate tools and define the protocols necessary for the introduction of these tools into the bacteria.
We offer the possibility of training members of the collaborative team, remotely or hosted on site for a few weeks/months.
2/ In partnership with CNRS Formation Entreprises, SIG.b offers training in cutting-edge technologies (in French):
• CRISPRi: an innovative application of CRISPR to modulate gene expression in bacteria
• Genome engineering combined with CRISPR to generate scar-free mutations in bacteria.
We may also offer customized in situ training (theoretical and/or practical).
Equipment
Know-how:
Modification of the bacterial genome and/or modulation of gene expression.
• Genome editing: point mutation, deletion, insertion (tag), by allelic exchange and/or CRISPR
• Genetic screening: isolation of mutants by selection or phenotypic identification (suppressor, synthetic lethality, etc.)
• Gene regulation: CRISPR interference, transcriptional activation, overexpression
• Bioinformatics: Identification of mutations, genome assembly post NGS sequencing
Biosafety laboratories:
Levels 1 – 2 – 3
E. coli strain collections:
• Gene inactivation (KEIO)
• Over expression (ASKA)
• Tagged genes (SPA tag)
Bacteria:
Escherichia coli, Salmonella Typhimurium
Mycoplasma
Mycobacteria
Staphylococcus aureus
Streptococcus pneumoniae, Group B Streptococcus Lactobacillus
etc.
Tools:
• Plasmids and phages for recombineering
with single or double-strand DNA
• CRISPR derived tools for single- or double-strand DNA cleavage (counter selection of original clones), specific addressing of accessory functions, base editing, etc.
• CRISPRi gRNA library
• Plasmid libraries for gene overexpression, Tn seq (Mariner)
Project 1
Description :
In 2019, SIG.b started a collaboration with the IEVA team (LMGM) aimed at identifying putative BAM-interactors. The Beta-barrel Assembly Machinery (BAM) is an essential complex that promotes the biogenesis of outer membrane proteins in Gram negative bacteria. A quantitative mass-spectrometry approach identified putative BAM-interactors.
Service Objectives:
To gain insights into the function of as yet uncharacterized BAM-interacting factors, a CRISPR interference (CRISPRi) screen was set up to define genes conferring a synthetic fitness defect. These experiments have been conducted by SIG.b in the Laboratory of “biologie de synthèse” (Institut Pasteur) which is having the expertise in high throughput CRISPRi silencing.
We validated our genome-scale approach by selectively targeting via CRISPRi a couple of the genes that were in the screen output and demonstrating that shutting down their expression leads to negative growth fitness.
Our experiments allow to highlight several genes of which further genetic and biochemical analysis showed that they are at the crossroad between outer membrane biogenesis and septal peptidoglycan hydrolysis. Such work not only allowed elucidation of the link between E. coli BAM-dependent pathway and major envelope remodelling processes but also the acquisition in LMGM of a CRISPRi-based screening method, nowadays so central in molecular and cellular microbiology.
Funding:
Costs (consumables and mission expenses) were covered by the collaborating team.
Associated publication:
Elife 2021; doi: 10.7554/eLife.67817. Ranava D, Yang Y, Orenday-Tapia L, Rousset F, Turlan C, Morales V, Cui L, Moulin C, Froment C, Munoz G, Rech J, Marcoux J, Caumont-Sarcos A, Albenne C, Bikard D, Ieva R. Lipoprotein DolP supports proper folding of BamA in the bacterial outer membrane promoting fitness upon envelope
Project 2
Description :
In 2021, SIG.b started a collaboration with the Giraud team from InTheRes (ENVT-INRAE Toulouse).
Ciprofloxacin is an antibiotic of the fluoroquinolone (FQ) family whose primary target is DNA gyrase. Repeated exposure of a susceptible strain, Escherichia coli ATCC25922 to ciprofloxacin at therapeutic concentrations led to isolation of two tolerant mutants. Whole Genome Sequencing (WGS) of the mutants revealed point mutations in the gyrB gene, one of the two subunits of DNA gyrase.
Service Objectives:
Strain Reconstruction by Site-Directed Mutagenesis
– ATCC25922 is the reference strain for antibiograms but is not a laboratory strain; its transformability is low and was improved.
– We reintroduced into the parental strain the mutations obtained by experimental selection in order to verify that these Single Nucleotide Polymorphisms are readily responsible for resistance and tolerance phenotypes.
Funding:
The flat-rate fee and consumables were financed by the INRAE team. The doctoral student from the INRAE team was hosted for 6 months in the service to learn and carry out genetic experiments.
Associated publication:
Front Microbiol. 2022; doi: 10.3389/fmicb.2022.908296. Perault A, Turlan C, Eynard N, Vallé Q, Bousquet-Melou A and Giraud E (2022). Repeated Exposure of Escherichia coli to High Ciprofloxacin Concentrations Selects gyrB Mutants That Show Fluoroquinolone-Specific Hyperpersistence.
Project 3
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.
– PLoS Pathog. 2023; doi: 10.1371/journal.ppat.1011437
– Front Microbiol. 2022; doi: 10.3389/fmicb.2022.908296
– Elife 2021; doi: 10.7554/eLife.67817
– PLoS One 2020; doi: 10.1371/journal.pone.0226472
Affiliation
