Cloning of RND Pump genes from B. Fragilis into an E. coli Vector
Mentor:Hannah Wexler, Professor of Medicine, University of California Los Angeles
B. fragilis is a leading cause for sepsis and other infections among humans undergoing a variety of traumas, mainly surgery. This gram negative bacterium resides in the human gut, where it has found the
anaerobic environment suitable for its needs. It is of interest, foremost, due to the threat it proposes to human life, a fact that is amplified further by the mechanisms it has developed against various antibiotics.
For instance, it, like many other bacteria, is highly resistant to penicillin.
A main feature allowing B. fragilis to proliferate as an infectious agent consists of a series of pumps found on its outer membrane. It is a very complicated and not well-understood feature of this bacterium, and is
also the focus of the research I will be conducting in Dr. Wexler’s lab. Generally, I will hope to aid in the further understanding of these mysterious bacteria via the research I do on 3 of the 16 pumps on B.
fragilis’ outer membrane. To elaborate, I will be cloning the RND pump genes called Bme 7, Bme 8, and Bme 9 into an E. coli vector, and hypothesize that the vector will accept these genes. Towards this end, I will use a variety of techniques such as primer design, PCR, ligation, transformation, DNA sequencing and all associated techniques
So far, I have been able to successfully clone Segments of these 3 different genes, with zero mutations or discrepancies. As a result, I have been able to provide my lab novel samples to include in their database for future studies which will prove indispensable with regards to the previously mentioned dangers of a B. Fragilis infection.
The hypothesis was, thus, correct in that I was able to put these genes into the Vector successively after forming well though-out experiments.