Difference between revisions of "Team:Queens Canada/Results"
| Line 54: | Line 54: | ||
<div style="background-color:#afbee8;color:black;font-weight:100;padding:50px 50px 50px 50px;line-height:150%;"> | <div style="background-color:#afbee8;color:black;font-weight:100;padding:50px 50px 50px 50px;line-height:150%;"> | ||
| − | <h1> | + | <h1>MhLap Dextran Binding</h1> |
| + | <br> | ||
| − | |||
<p>The first step in assessing the feasibility of using dextran to crosslink M. hydrocarbonoclasticus to our biofilm was to test the ability of this bacterium’s sugar binding domain to bind dextran. Luckily, Tyler Vance of Dr. Davies’ lab already had the sugar-binding MhLap Region III cloned into pET-28 for overexpression in E. coli. We expressed, then purified this construct using a Ni-NTA column. Purified MhLap Region III fractions were pooled. | <p>The first step in assessing the feasibility of using dextran to crosslink M. hydrocarbonoclasticus to our biofilm was to test the ability of this bacterium’s sugar binding domain to bind dextran. Luckily, Tyler Vance of Dr. Davies’ lab already had the sugar-binding MhLap Region III cloned into pET-28 for overexpression in E. coli. We expressed, then purified this construct using a Ni-NTA column. Purified MhLap Region III fractions were pooled. | ||
We then set up a simple affinity chromatography binding assay. A Pasteur pipette was filled half-way with sephadex G-200 slurry. Purified MhLap was then added, followed by five washes with buffer. We then washed twice with 5 mg/mL dextran solution. Finally, the column was washed twice with EDTA to nonspecifically elute all protein. The collected fractions can be seen in the following SDS gels:</p> | We then set up a simple affinity chromatography binding assay. A Pasteur pipette was filled half-way with sephadex G-200 slurry. Purified MhLap was then added, followed by five washes with buffer. We then washed twice with 5 mg/mL dextran solution. Finally, the column was washed twice with EDTA to nonspecifically elute all protein. The collected fractions can be seen in the following SDS gels:</p> | ||
<img src="https://static.igem.org/mediawiki/2017/6/61/T--Queens_Canada--DextranGel.png"> | <img src="https://static.igem.org/mediawiki/2017/6/61/T--Queens_Canada--DextranGel.png"> | ||
| + | <br> | ||
| + | <br> | ||
<figcaption><center><b>Fig 1. MhLap dextran binding experiment gels.</b> From left to right, the lanes contain flow through (FT), buffer washes 1-5, dextran (5 mg/mL) washes 1-2, and 1M EDTA washes 1-2. </font></center></figcaption> | <figcaption><center><b>Fig 1. MhLap dextran binding experiment gels.</b> From left to right, the lanes contain flow through (FT), buffer washes 1-5, dextran (5 mg/mL) washes 1-2, and 1M EDTA washes 1-2. </font></center></figcaption> | ||
Revision as of 17:18, 23 October 2017
MhLap Dextran Binding
The first step in assessing the feasibility of using dextran to crosslink M. hydrocarbonoclasticus to our biofilm was to test the ability of this bacterium’s sugar binding domain to bind dextran. Luckily, Tyler Vance of Dr. Davies’ lab already had the sugar-binding MhLap Region III cloned into pET-28 for overexpression in E. coli. We expressed, then purified this construct using a Ni-NTA column. Purified MhLap Region III fractions were pooled. We then set up a simple affinity chromatography binding assay. A Pasteur pipette was filled half-way with sephadex G-200 slurry. Purified MhLap was then added, followed by five washes with buffer. We then washed twice with 5 mg/mL dextran solution. Finally, the column was washed twice with EDTA to nonspecifically elute all protein. The collected fractions can be seen in the following SDS gels:
