"
Team:Cambridge/Project/In Vitro
From 2011.igem.org
(Difference between revisions)
(→Objective Two - Purify reflectin in vitro) |
(→Objective Two - Purify reflectin in vitro) |
||
| Line 1: | Line 1: | ||
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}} | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}} | ||
| - | =Objective Two - Purify | + | =Objective Two - Isolate and Purify Reflectin ''in vitro''= |
| + | |||
| + | Purification of recombinant reflectin from bacteria was necessary in order to perform all of the subsequent ''in vitro'' studies of the protein. An over-exressing vector construct was used to maximise yield, and a his-tag was used for purification purposes. | ||
[[File:camflow2.jpg| centre | 600px]] | [[File:camflow2.jpg| centre | 600px]] | ||
| - | ==Tagging reflectin== | + | ==Tagging the reflectin gene== |
| + | |||
| + | The reflectin genes were his-tagged by incorporating the his sequence into the primers used to clone the gene. His tagging was chosen because... | ||
| + | |||
| + | ==Overexpression== | ||
| - | + | We chose to use a high copy plasmid with a pBAD promotor in order to overexpress reflectin. This produces inclusion bodies, which will create unfolded reflectin, but this is not a concern because the solvents | |
| - | == | + | ==Protein extraction and purification== |
Revision as of 17:08, 15 August 2011
Loading...
Contents |
Objective Two - Isolate and Purify Reflectin in vitro
Purification of recombinant reflectin from bacteria was necessary in order to perform all of the subsequent in vitro studies of the protein. An over-exressing vector construct was used to maximise yield, and a his-tag was used for purification purposes.
Tagging the reflectin gene
The reflectin genes were his-tagged by incorporating the his sequence into the primers used to clone the gene. His tagging was chosen because...
Overexpression
We chose to use a high copy plasmid with a pBAD promotor in order to overexpress reflectin. This produces inclusion bodies, which will create unfolded reflectin, but this is not a concern because the solvents
