Genetics and Evolution of Microbial Secondary metabolism

Strain improvement will be pursued (depending on project goals) in one of the following ways (or in combination):

• selection of spontaneous overproducing variants
• transposon mutagenesis
• overexpression of pathway-specific or global regulatory genes
• introduction in the genome of strain of interest of rpsL alleles known to increase antibiotic production

We have all necessary instrumentation for microbiological and molecular genetic work.

Our requirements:

• initial level of production (mg/L in defined liquid medium)
• method of antibiotic quantification (simple bioassay- or TLC- or colorimetry-based methods)
• sequence of the gene cluster responsible for production of antibiotic of interest.

If the initial strain is wild type isolate (not selected/improved previously), we deliver its derivatives producing at least two times more antibiotic than the wild type. In most cases we expect to generate these derivatives within one year or less.

We develop methods for introduction of replicative and integrative plasmid DNA into the cells of actinomycetes.

We offer expertise in four approaches:

• electroporation
• intergeneric conjugation
• protoplast transformation
• transformation of competent cells.

We first test the most efficient method (conjugation), and only proceed to other approaches if this is unsuccessful. Per customer request we can use a different experimental scheme.

We have a large suite of actinomycete vectors that span all major taxa of this order; we can also test plasmids provided by the customer; he(she) also may request to deliver specific genes. We require: strain with known genus affiliation.

Deliverables
We deliver step-by-step description of the gene transfer procedure and provide recombinant strains harboring plasmids that were successfully introduced into the strain. In most cases we deliver these materials within six months.

We will develop a cultivation conditions for actinomycetes - for sporulation or production of antibiotics