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Team:Sheffield/Lipase
From 2014.igem.org
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<h2>Lipase </h2> | <h2>Lipase </h2> | ||
| - | <p> | + | <p>The image to the left is taken on a transilluminator to show the results of our main lipase assay.</p> |
<br> | <br> | ||
| - | <p>Lipases are generally assayed using tween 80 in combination with Nile blue, but this has been shown to react to esterases too. When incorporated into a plate, rhodamine B acts as a | + | <p>Lipases are generally assayed using tween 80 in combination with Nile blue, but this has been shown to react to esterases too. When incorporated into a plate, rhodamine B acts as a reporter that shows breakdown of lipids in the suspension. By making plates containing both olive oil and rhodamine, we were able to check for lipase activity by placing plates under a UV light in the transilluminator.</p> |
<br> | <br> | ||
| - | <p>The | + | <p>The plate shows 10μl of supernatant from either BBa_J23110 or “Lip9” (our Lipase construct containing a the BBa_J23110 promoter and a second plasmid encoding the ABC transporter). A zone of fluorescence can clearly be seen around the spot at which the supernatant was pipetted on. The promotor vector supernatant has acted as a negative control to show that the zone of fluorescence is indeed caused by our lipase construct, and not the cells. </p> |
<br> | <br> | ||
| - | <p> | + | <p>In this second image, the same plate is shown in normal lighting conditions. A zone of colour change can be seen where the negative control has been added, but this can’t be seen under the UV light. It appears that the lipase has turned the rhodamine from pink to a bright orange. </p> |
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 00:26, 18 October 2014
Results
Lipase
The image to the left is taken on a transilluminator to show the results of our main lipase assay.
Lipases are generally assayed using tween 80 in combination with Nile blue, but this has been shown to react to esterases too. When incorporated into a plate, rhodamine B acts as a reporter that shows breakdown of lipids in the suspension. By making plates containing both olive oil and rhodamine, we were able to check for lipase activity by placing plates under a UV light in the transilluminator.
The plate shows 10μl of supernatant from either BBa_J23110 or “Lip9” (our Lipase construct containing a the BBa_J23110 promoter and a second plasmid encoding the ABC transporter). A zone of fluorescence can clearly be seen around the spot at which the supernatant was pipetted on. The promotor vector supernatant has acted as a negative control to show that the zone of fluorescence is indeed caused by our lipase construct, and not the cells.
In this second image, the same plate is shown in normal lighting conditions. A zone of colour change can be seen where the negative control has been added, but this can’t be seen under the UV light. It appears that the lipase has turned the rhodamine from pink to a bright orange.
