Cambridge/2008/Turing Pattern Formation/Promoters and Vectors: Difference between revisions

Revision as of 10:58, 24 July 2008

The IPTG-Pspac system demonstrates efficient and titratable induction of bGal expression.

New integration vector for high level, constitutive expression of cloned inserts
- From Bacillus Genetic Stock Center: Dr. Brian Jester of Trinity College, Dublin, Ireland has kindly donated a novel vector, pBCJ164.3, to our collection. The plasmid contains the 5' and 3' ends of the Bacillus subtilis rpsD gene, together with its promoter and transcription terminator. An NdeI site within this cassette allows for inserted fragments to be placed under the control of the strong rpsD promoter. Like other integration vectors, pBCJ164.3 replicates in E. coli but not in B. subtilis. When a recombinant plasmid is isolated from E. coli and transformed into a recombination-proficient B. subtilis host with selection for chloramphenicol resistance, a non-mutagenic Campbell-type insertion even should take place within the host chromosomal rpsD locus.

New ectopic integration vectors for Bacillus subtilis
- From tbhe Bacillus Genetic Stock Center:Rebecca Middleton of the University of California, Berkeley, has generously donated to the BGSC a set of novel integration vectors. The vectors integrate into the Bacillus subtilis chromosome “ectopically,” that is, at a locus targeted by homologous sequences within the vector itself, rather than by sequences within a cloned insert. Each vector contains an integration cassette consisting of the 5’ and 3’ ends of a non-essential chromosomal gene, interrupted by a selectable antibiotic resistance marker and a multiple cloning site. When the vectors are introduced into a host strain by transformation with selection for antibiotic resistance, a double-crossover event replaces the chromosomal locus with the plasmid-borne cassette, including any fragments that have been inserted into the cloning sites. The six plasmids within the collection allow the user to target any of three loci—gltA, pyrD, or sacA—with selection for either kanamycin or chloramphenicol resistance. The collection also includes six control strains in which the cassettes, without inserts, have been integrated into the chromosomal loci.

This vector from BGSC can test different promoters, but it needs a special Bacillus strain that has an ERM site where amyE goes normally:
Can test beta-glactosidase activity
On page 25 of the BGSC catalog

@@ Line 25: / Line 25: @@
 *[http://www.bgsc.org/NewProducts/pHCMC.pdf This shuttle vector does not integrate, but is stable enough]
-===pBCJ164===
+===pBCJ164, strong iPTG-induced integration vector===
 * [http://www.bgsc.org/NewProducts/pBCJ164.pdf New integration vector for high level, constitutive expression of cloned inserts]
 ** ''From Bacillus Genetic Stock Center:'' Dr. Brian Jester of Trinity College, Dublin, Ireland has kindly donated a novel vector, pBCJ164.3, to our collection.  The plasmid contains the 5' and 3' ends of the Bacillus subtilis rpsD gene, together with its promoter and transcription terminator.  An NdeI site within this cassette allows for inserted fragments to be placed under the control of the strong rpsD promoter.  Like other integration vectors, pBCJ164.3 replicates in E. coli but not in B. subtilis.  When a recombinant plasmid is isolated from E. coli and transformed into a recombination-proficient B. subtilis host with selection for chloramphenicol resistance, a non-mutagenic Campbell-type insertion even should take place within the host chromosomal rpsD locus.