Welcome to the Vector Tools Site
This web site contains vector maps for a variety of cloning vectors (largely yeast based) that Rob Shanks, a former post-doc in the lab, developed to facilitate studies in a broad range of Gram-negative organisms. Because of the large number of vectors generated, and a lack of resources to distribute them, we have arranged for Presque Isle Cultures to distribute E. coli strains carrying the vectors for a nominal fee to academic labs. Companies should contact George O’Toole directly at georgeo@Dartmouth.edu.
PUBLISHED VECTOR TOOLS
Plasmid maps for the vectors published recently in Applied Environmental Microbiology can be downloaded below:
Reference : Shanks, R.M.Q., N.C. Caiazza, S.M. Hinsa, C.M. Toutain, and G.A. O'Toole. 2006. A Yeast-Based Molecular Tool Kit for Manipulation of Gram-Negative Bacterial Genes, Appl. Environ. Microbiol. In press.
UNPUBLISHED VECTOR TOOLS
We have also developed several new vectors that are not yet published but are available for distribution. These vectors all are able to replicate in Saccharomyces cerevisiae for cloning via yeast homologous recombination, but all can be used as standard vectors.
Expression vectors: pMQ122 and pMQ123 incorporate Ptac/lacI Q promoters with the ColE1 (pUC) and pRO1600 replicons allowing for stable maintenance in enterobacteria, pseudomonads and other Gram-negative species. pMQ124 and pMQ125 expression vectors have the P BAD /araC promoter system with either the ColE1 (pUC) or p15a replicon for high or low-copy maintenance in enterobacteria and the pRO1600 replicon for broad host range replication. Additionally pMQ124 and pMQ125 have lacZ a under control of P BAD for blue-white screening.
Shuttle vectors: All these have origins of conjugal transfer and Plac driving lacZ a expression, and can be used for blue white screening or constitutive expression of desired genes. pMQ113 incorporates a temperature sensitive version of the Lactococcus lactus pWV01 replicon that is capable of replication in both Gram-positive and Gram-negative bacteria. This plasmid can be used as a temperature-sensitive suicide vector for many purposes in a wide range of organisms. pMQ117 has the Salmonella -derived pSC101 origin of replication that is useful for expression in enterobacteria and for maintenance of very large inserts. pMQ117 is a temperature sensitive plasmid and is compatible with a number of other replicons, so it is useful as a conditional suicide vector or for multi-plasmid applications. pMQ131 and pMQ132 replicate in a very broad range of Gram-negative bacteria due to the pBBR1-derived replicon, originally from a cryptic Bordetella bronchiseptica plasmid. These plasmids are maintained at medium copy number in a number of bacteria including the pseudomonads and are compatible with a wide variety of plasmid replicons. They differ by selectable marker (see maps below).
Allelic replacement vector: pMQ118 uses a plasmid R6K origin for pi-dependent replication, and thus is useful as a suicide vector in most Gram-negative bacteria. This plasmid also has an rpsL gene from E. coli for streptomycin-counter selection in certain strain backgrounds making it a useful allelic-replacement vector. This plasmid also has Plac-lacZ a and an oriT.
Reference : There is no reference for these vectors at this time. Acknowledging R.M.Q. Shanks and G.A. O'Toole will be fine.
The accession numbers for the vectors are:
The accession numbers for the unpublished vectors are:
*Glossary of terms on plasmid maps:
oriT – origin of conjugal transfer
ColE1 – high-copy version of ColE1 replicon, for replication in E. coli and other enterobacteria
sacB - Counter-selectable marker, conferrs sucrose sensitivity
Plac-lacZ a - Consitutive expression in lacI negative strains, lacZ a has a multicloning site, useful for blue white screening, or replace lacZ a with your gene using yeast recombination
p15a - Lower copy replicon for E. coli and other enterobacteria
pRO1600 - Lower copy replicon for pseudomonads, Klebsiella , and possibly other Gram-negatives
P BAD - L-arabinose inducible promoter system, includes the araC regulator
P BAD -lacZ a – P BAD driving expression of lacZ a with a multicloning site for arabinose-dependent blue-white screening
P BAD - gfp - P BAD driving expression of GFP-mut3 , a bright and stable varient of GFP . For translational and transcriptional GFP -fusions. GFP can also be completely replaces by tradional or yeast in vivo cloning.
P tac - gfp - P tac driving expression of GFP-mut3 , a bright and stable varient of GFP . For translational and transcriptional GFP -fusions. GFP can also be completely replaces by tradional or yeast in vivo cloning. Ptac is capable of very high levels of expression and is inducible using IPTG. Ptac -expresssion is known to be somewhat leaky in the absence of inducer.
lacI Q - Negative regulator of Ptac in the absence of inducer.
RK2 - The minimal RK2 replicon. Incompatability group P. Replicates at low copy numbers in over thirty species of Gram-negative bacteria.
pSC101 - Low copy replicon for enterobacteria. Compatible with ColE1, p15a, RK2, pBBR1 and other replicons. This version is unable to efficiently replicate at 37°C or above and can thus be used as a conditional suicide-vector.
pWV01 - Low copy replicon for Gram-positive and Gram-negative bacteria. Known to replicate in E. coli , Staphylococcus sp , Streptococcus sp , Lactococcus sp . and probably others. This verion is temperature sensitive, such that cells with this plasmid should be grown at room-temperature to 30°C. This replicon has been used for allelic replacement, transposon delivery systems, and as a shuttle vector.
The orfB, orfD, repA and repC proteins are from pWV01.
pBBR1 - Medium copy replicon for Gram-negative bacteria. Has a very broad host range and is compatible with a wide variety of plasmids.
rpsL - E. coli rpsL gene, codes for ribosomal protein S12. rpsL confers sensitivity to streptomycin, and can be used as a counterselectable marker.
aacC1 - Gentamycin resistance determinant.
T1T2 - Terminators from rrnB from E. coli.
bla - Ampicillin resistance determinant
aphA - 3 - Kanamycin resistance determinant, useful in Gram-positive and Gram-negative bacteria
kanMX - Kanamycin resistance determinant, useful in Gram-negative bacteria, does not confer kanamycin resistance to Staphylococcus aureus , does confer G418-sulfonate resistance to Saccharomyces cerevisiae .
PoriV - same as pRO1600 above
URA3 - Complements uracil auxotrophic phenotype Saccharomyces cerevisiae strains with ura3 mutations.
CEN/ARSH - Low copy yeast replicon
2µ - High copy yeast replicon