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Team:Dundee/Parts/Ourbiobricks
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| + | <h2>Success and Progress</h2> | ||
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| + | <h2>TorA signal sequence works!</h2> | ||
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| + | <p>The combination of the signal sequence of TorA with PP1-Ha proved worth our time and effort, as when we carried out cell fractionation on cells expressing this construct, and carried out Western Blots of the fractions, we discovered that PP1 was being transported to the periplasm of <i>E.coli</i>!</p> | ||
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| + | <h2>MalE lodged in the inner membrane of <i>E.coli</i>!</h2> | ||
| + | <p>By cell fractionation and Western Blot methods, we found out that MalE signal sequence joined to PP1-HA became stuck in the inner membrane of E.coli cells, therefore wouldn’t be available to bind microcystin from the cell surroundings.</p> | ||
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Revision as of 14:12, 15 August 2013
Our Biobricks
What are our Biobricks?
We submitted 7 constructs to the Registry of Standard Biological Parts, related to our biological mop system for the removal of microcystin from freshwater.
Here they are:
- The signal sequence of the MalE gene.
- The signal sequence of the PrsA gene.
- The signal sequence of the TorA gene.
- The PP1 (protein phosphatase 1) gene with a HA tag attached immediately downstream.
- The signal sequence of the MalE gene, with PP1-HA attached immediately downstream.
- The signal sequence of the PrsA gene, with PP1-HA attached immediately downstream.
- The signal sequence of the TorA gene, with PP1-HA attached immediately downstream.
*The HA tag was used for Western Blotting.
All of our inserts and the iGEM vector pSB1C3 were digested at PstI and EcoRI restriction sites and then ligated together to produce our BioBricks.
Inserts and iGEM vector
Fig.1. Diagram showing the different microcystin mop BioBricks made by the team.
The Theory Behind Our Biobricks
As we were looking into different methods of transportation of PP1 to the cell membrane, we considered both the TAT and SEC protein targeting pathways.
TAT pathway ( E.coli )
The TAT pathway is a biological method for the transport of proteins into the cell membrane, with the protein being folded before entry to the plasma membrane. The protein TorA is an example of such proteins controlled by TAT. We combined the signal sequence of the TorA gene with PP1-HA in order to transport protein phosphatase 1 to the cell membrane; in the hope that it would anchor there and mop up microcystin from the cell surroundings.
SEC pathway ( E.coli and B.subtillus )
The SEC pathway is another biological method for integrating proteins into the cell membrane, with the protein being folded as it enters the plasma membrane. MalE and PrsA are both proteins destined to form a union with the cell membrane by the SEC technique. By each combining the signal sequences MalE and PrsA with PP1-HA separately, we had the same aim for the transport of PP1 within the cell after production, as with TorA in the TAT pathway.
Success and Progress
TorA signal sequence works!
The combination of the signal sequence of TorA with PP1-Ha proved worth our time and effort, as when we carried out cell fractionation on cells expressing this construct, and carried out Western Blots of the fractions, we discovered that PP1 was being transported to the periplasm of E.coli!
MalE lodged in the inner membrane of E.coli!
By cell fractionation and Western Blot methods, we found out that MalE signal sequence joined to PP1-HA became stuck in the inner membrane of E.coli cells, therefore wouldn’t be available to bind microcystin from the cell surroundings.
