The TonB-ExbB-ExbD complex is essential for the siderophore mediated acquisition of iron by Gram negative bacteria. The system provides energy from the proton motive force to the outer membrane in order for the iron siderophore complex to enter into the cell. The main protein involved in energy transduction, TonB, has been extensively studied in the species Escherichia Coli. It has been determined that the protein consists of 239 amino acids. In comparison, however, the TonB of Rhizobium leguminosarum consists of 457 amino acids with the same conserved regions. What is in question, therefore, is how the additional amino acids effect the structure of the C-terminal region of the protein and how such information can give insight into the way in which the proton motive force functions to provide energy to the outer membrane receptor. The protein regions of R. leguminosarum TonB chosen for study were 120 and 248 amino acids from the C-terminal end. Genomic DNA was isolated, primers were designed for each fragment, and polymerase chain reactions were performed. After appropriate restriction enzyme digestion, each DNA fragment was ligated into the plasmid pET-17b and then transformed into Escherichia Coli BL21 (DE3). Successful transformation of the 120 amino acid fragment was followed by expression via IPTG induction & extraction of protein. Afterwards, a T7-tag affinity column was attempted to collect the protein for analysis; however, a sufficient amount of protein was not eluted. The procedure will be repeated for obtaining sufficient protein for crystallization or NMR spectrometric analysis.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:honors-1421 |
| Date | 01 May 2017 |
| Creators | Baxter, Brooke E |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Undergraduate Honors Theses |
| Rights | Copyright by the authors., http://creativecommons.org/licenses/by-nc-nd/3.0/ |
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