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Team:Cornell/project/wetlab/results/protein
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<h3>Western Blots for MtrB</h3> | <h3>Western Blots for MtrB</h3> | ||
| - | To assess translational efficiency of | + | |
| + | To assess the translational efficiency of MtrB in our constructs, we have | ||
| + | begun western blots for MtrB. With these experiments, we hope to correlate the | ||
| + | sensitivity of our constructs to changes in analyte concentration. In addition, we | ||
| + | would like to determine the amount of leaky expression in our constructs that may | ||
| + | account for oversaturation of mtrB and thus, loss of sensitivity to increasing analyte. | ||
| + | This is particularly relevant for our Salicylate constructs which we hypothesize are | ||
| + | unresponsive to Salicylate because the Mtr complex is being overwhelmed by MtrB. | ||
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| - | + | To detect MtrB, we decided to use two approaches. First, we decided to use | |
| - | + | the commonly used cell biological method of appending poly-his tags for detection | |
| - | + | by poly-His antibodies. We are in the process of separately adding poly His tags | |
| - | + | with short Gly-Ser-Gly linkers to the N and C termini of mtrB in p14K, p16K, SAL, | |
| - | + | and SAL2 for a total of 8 potential biobricks. We currently have 5 of the 8: P14K C- | |
| - | + | His, P16 N-His, Sal C-His, Sal2 N-His, and Sal2-CHis <a href="https://2012.igem.org/Team:Cornell/project/wetlab/results/biobricks">(See our biobrick page)</a> . We believe these constructs | |
| - | + | may also be used for in vitro characterization of mtrB, and will also facilitate the | |
| - | + | purification of mtrB to obtain its crystal structure. Our second approach will be to | |
| - | + | detect MtrB using antibodies from Dr. Liang Shi from Pacific Northwest National | |
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Latest revision as of 01:22, 27 October 2012
MtrB Protein Expression
Western Blots for MtrB
To assess the translational efficiency of MtrB in our constructs, we have begun western blots for MtrB. With these experiments, we hope to correlate the sensitivity of our constructs to changes in analyte concentration. In addition, we would like to determine the amount of leaky expression in our constructs that may account for oversaturation of mtrB and thus, loss of sensitivity to increasing analyte. This is particularly relevant for our Salicylate constructs which we hypothesize are unresponsive to Salicylate because the Mtr complex is being overwhelmed by MtrB.To detect MtrB, we decided to use two approaches. First, we decided to use the commonly used cell biological method of appending poly-his tags for detection by poly-His antibodies. We are in the process of separately adding poly His tags with short Gly-Ser-Gly linkers to the N and C termini of mtrB in p14K, p16K, SAL, and SAL2 for a total of 8 potential biobricks. We currently have 5 of the 8: P14K C- His, P16 N-His, Sal C-His, Sal2 N-His, and Sal2-CHis (See our biobrick page) . We believe these constructs may also be used for in vitro characterization of mtrB, and will also facilitate the purification of mtrB to obtain its crystal structure. Our second approach will be to detect MtrB using antibodies from Dr. Liang Shi from Pacific Northwest National Laboratory.
