Cambridge/2008/Turing Pattern Formation/Promoters and Vectors

Ordering from BGSC

ECE171 kan, pyrD ECE172 cat, gltA ECE147 (pMUTIN-cMYC) ECE 112 (spoVG-lacZ reporter, insert at amyE) ECE153 (xylose inducible gfp using Pspac)

ECE149, ECE150, ECE151, ECE162

pAD123, 43-25: ECE166 ECE165

ECE188,189,190 (pMCHC set) ECE176 (rpsD promoter, non-mutagenic insertion)

1A771 - erm insertion at amyE locus

Episomal Vectors

• pHCMC02,pHCMC04,pHCMC05 - Expression Vectors
  • Original Publication
  • one is IPTG-inducible, one is xylose-inducible, and one is weakly constitutive

• PAD123 and PAD43-25 - GFP (fusion) vectors w/ and w/o const. promoters
  • one has a B. cereus Pupp promoter, which apparently is very strong and works in B. subtilis

Integrating Vectors

• pLacZ - lacZ reporter
  • integrating vector at amyE locus w/ lacZ transcriptional fusion, for promoter assay

• pBCJ164.3
  • integration vector, rspD site, uses rspD promoter; "extreme" expression, may cause deleterious effects for some products

• pMUTIN-cMYC - antibody tagging vector
  • inserts into specified chromosomal site, adds antibody tag for protein purification

• pSG1164/pMUTIN-GFP+
  • c-terminal GFP fusion plasmid, inducible with xylose/IPTG, insert at any amyE/any point

• pMUTIN4

• aiiA knockout if they have it

• pDG1728
  • test promoters

• need strain 1A771 for amyE recombination with ERM?

• protease knockout? BW600,700,800?

Promoters

• The Pspac promoter is tightly regulated by iPTG

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

More info on Vectors

PAD43-25

• This vector constitutively expresses GFP using the Pupp promoter:

Gram-Positive E. coli Shuttle Vector

• no messing about required, unless you want to do a GFP fusion

PnZ8901

• This is the shuttle vector from last year

pNZ8901 shuttle vector

pHCMC - stable non-integrating shuttle vector

• Most shuttle vectors are unstable in gram-positive bacteria, but this one is more stable
• Could be an alternative to integration-based vectors
• 3 flavors, iPTG-inducible Pspac, xylose-inducible, weak constiuitive lepA
• pHCMC pdf from BGSC
• Plasmid paper describing their use!

pBCJ164, strong iPTG-induced integration vector

• 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.

ectopic integration vectors at different loci

• 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.

pDG1662 promoter test vector

• This vector from BGSC can test different promoters,
• 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

pDG1662 integration vector