MYCOBACTERIA FUNCTIONAL SCREENING AND/OR EXPRESSION VECTORS

• Main Authors: TIMM, JULIANO, GICQUEL, BRIGITTE, LIM, ENG MONG, BERTHET, FRANCOIS-XAVIER, PORTNOI, DENIS
• Format: Patent
• Language: English; French; German
• Published: 13.02.2008
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; COMPOSITIONS THEREOF; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; CULTURE MEDIA; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; HUMAN NECESSITIES; HYGIENE; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; MEDICAL OR VETERINARY SCIENCE; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; ORGANIC CHEMISTRY; PEPTIDES; PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES; PROCESSES OF PREPARING SUCH COMPOSITIONS; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

New recombinant screening, cloning and/or expression vectors (V1 and V2), able to replicate in mycobacteria contain: (i) a replicon functional in mycobacteria; (ii) a selection marker; (iii) a reporter cassette comprising: (a) a multiple cloning site (polylinker, PL); (b) a transcription terminator active in mycobacteria (pref. coliphage T4 terminator) and located upstream of PL, and for vector V1: (c) a nucleotide sequence (ntseq) derived from a gene coding for an expression marker, or protein export and/or secretion marker, the ntseq lacking an initiation codon and regulation sequences; or for vector V2: (c') a reporter gene whose expression can be analysed in the presence of different regulatory sequences. Pref. vector V1 further comprises a ntseq (A) from a mycobacterium, cloned into PL. In a new screening method, ntseq (A), obtained by restriction enzyme digestion or by in vitro amplification, is inserted into the PL of vector V1 and the resulting vector is opt. replicated before being used for transformation of host cells. DNA is isolated from positive transformants (i.e. cells expressing the export/secretion marker) and subcloned. Inserts from the subclones are isolated to allow characterisation of the mycobacteria sequences they contain. Ntseqs isolated by this method are claimed, per se.