Genetic tools

One of my interests as a molecular microbiologist has been the design and construction of genetic tools.  Below are descriptions of three publications detailing plasmid vectors designed for several different applications.  For detailed viewing of the plasmid maps, I recommend CLC sequence viewer which is free to download.

pHERD series vectors: Controlled expression from the PBAD promoter.

pHERD20T

The pHERD vectors are multi-copy vectors that are compatible with a wide range of bacteria.  Genes can be cloned into the multiple cloning site.  The PBAD promoter is repressed in the absence of arabinose.  Upon addition of arabinose to the growth medium the gene will be expressed and the level of expression correlates to the amount of arabinose supplied.  These vectors are useful for complementation studies,  overexpression of genes, expression for purification, and etc.  Click here for a complete list of studies which have used pHERD vectors.  If you are interested in obtaining the pHERD vectors for your research, please contact Dr. Hongwei Yu of Marshall University School of Medicine

Click to download .CLC files of the pHERD-vectors


pTJ1: Controlled PBAD gene expression from a single copy on the chromosome

pTJ1

By inserting gene expression constructs into the chromosome, the researcher can eliminate the need to maintain the plasmid with antibiotics which can sometime affect various aspects of an experiment.  To achieve this, we moved the araC and PBAD promoters from pHERD over into the pUC18-mini-Tn7T series vectors and named the vector pTJ1.  TJ stands for Thomas Jefferson, the founder of UVa and author of the Declaration of Independence.   Due to his curiosity we think Thomas Jefferson would have been very excited to see where science is today and named this vector in his honor.  The mini-Tn7 based pTJ1 encodes a mobile element which can be used to shuttle the construct directly to the chromosome.  Another advantage of pTJ1 is since only one copy of the construct is on the chromosome versus many copies, theoretically lower expression levels can be tested.   If you are interested in obtaining pTJ1 for your research, please contact Dr. Joanna Goldberg of Emory University.

Click to download .CLC file of pTJ1


mini-Tn7 based lux vectors for bioluminescent detection and expression analysis

pUC18T-mini-Tn7T-lux-Tp

Bioluminescence can be useful for both tracking the location of bacteria and also examining when promoters are activated/repressed.  The lux operon encodes products that when expressed, cause the bacteria to emit light.  This light emission can be measured by a number of different methods.  The mini-Tn7 lux vectors are built off of the same series of vectors as pTJ1.  We constructed the on of the versions with trimethoprim resistance due to the fact the PAO1 and PA14 mutant libraries use tet and gent resistance markers.     If you are interested in obtaining the lux vectors for your research, please contact Dr. Joanna Goldberg of Emory University.

Click to download .CLC files of the pUC18T-lux vectors