Formation of EzrA for Protein Expression
and in vitro FtsZ Polymerization
Assembly
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Cristina Fernandez |
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The Levin Lab seeks to define the regulatory networks governing bacterial cell division. Cytokinesis in most organisms begins with the formation of a cytoskeletal ring at the nascent division site. In bacteria this ring is essentially composed of the protein FtsZ, which is short for Filamenting temperature sensitive Z. As the cell divides, the ring on FtsZ constricts until division is complete and it disperses throughout the new cell's cytoplasm. Many factors govern FtsZ ring formation to ensure the cell divides medially.
My project focuses on creating a mutant version of the protein EzrA, Extra z rings A, a negative regulator that prevents FtsZ from forming rings at the poles of the cell. When EzrA is not present during the onset of cytokinesis, FtsZ rings tend to form at both poles of the cell as well as midcell. To test EzrA activity in vitro, Dr. Petra Levin and Rachel Schwartz developed an assay to make FtsZ polymers from monomers in a test tube, simulating natural FtsZ ring polymerization during cytokinesis. In this assay FtsZ polymers are pelleted by spinning the tube at 280,000 x g. Monomers stay in the supernatant. When the full length version of EzrA was added to this reaction polymerization was prevented and more FtsZ ended up in the monomer form in the supernatant. When a mutant version of EzrA missing the first two-thirds of the protein was added, most of the FtsZ remained in polymer form in the pellet. My project is to make a mutant piece of the first half of EzrA with the original start codon and a constructed stop codon. My objective for the summer was to clone the mutant ezrA into the pBAD/Thio-TOPO plasmid and transform it into E. coli. This plasmid will allow me to make EzrA protein by adding arabinose to an E. coli culture. Arabinose turns on the PBAD promoter in front of ezrA RNA that will then be translated to make EzrA protein.
I began my experiment by polymerase chain reaction (PCR) amplifying the ezrA gene flanked by the forward primer PL 99 and the reverse primer PL 120. The PCR product was then cloned into pBAD/Thio-TOPO vector and transformed into E. coli. To ensure that the PCR amplified EzrA was in fact inserted in the pBAD/Thio-TOPO vector, I did restriction enzyme double digests and single digests. If my PCR insert were in the pBAD/Thio-TOPO plasmid I would expect BamHI to cut the pBAD/Thio-TOPO plasmid in two places making three fragments of 436, 3753, and 4528 base pairs. PstI would cut the PCR insert once and linearize the plasmid. With the double digest I would expect to get three fragments of 3753 bp, 775 bp, and 436 bp. However, after running out the double digests on 1% agarose gels I got a band of approximately 4000 bp and 400 bp but nowhere else. This showed that BamHI was cutting, but PstI was not. The vector was religating without the PCR insert. We believe the pBAD/Thio-TOPO vector is bad and we have ordered a new kit with a new vector of a different lot number. When I can get a successful cloning of the PCR insert into the pBAD/Thio-TOPO vector, then I can check for protein expression in E. coli using assays.
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Natural Sciences Learning Center
Washington University - Biology
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