"
Team:Imperial College/Electron microscopy
From 2013.igem.org
Margarita K (Talk | contribs) |
Margarita K (Talk | contribs) |
||
| Line 3: | Line 3: | ||
<p> A way of showing that a degradation enzyme is functional is to incubate it with the polymer in question and scan the surface after some time. This is a widely used method for studying plastic degradation and we have taken advantage of it for characterising Proteinase K ([http://parts.igem.org/Part:BBa_K1149008 BBa_K1149008]) and observed how it "chews" on a PLA cup. </p> | <p> A way of showing that a degradation enzyme is functional is to incubate it with the polymer in question and scan the surface after some time. This is a widely used method for studying plastic degradation and we have taken advantage of it for characterising Proteinase K ([http://parts.igem.org/Part:BBa_K1149008 BBa_K1149008]) and observed how it "chews" on a PLA cup. </p> | ||
| + | <p> PLA can spontaneously hydrolyse in water. However, this only happens very slowly and at higher temperatures. Here is an SEM image from the literature (see reference below) where PLA was incubated for 9 and 20 days in deionized water at 58 °C. We are expecting our enzymes to have at least as visible effect in 3 or less days.</p> | ||
| + | <br> | ||
| + | https://static.igem.org/mediawiki/parts/2/2d/PLA_hydrolisis_SEM.jpg | ||
| + | <h5>method</h5> | ||
We cut small pieces of the cup and washed them with ethanol and deionised water in order to remove any potential contamination. | We cut small pieces of the cup and washed them with ethanol and deionised water in order to remove any potential contamination. | ||
Next, the substrates were incubated with cell lysate for 1, 2 and 3 days. The negative control was treated with cell lysate from MG1655 E.coli strain containing the empty vector. | Next, the substrates were incubated with cell lysate for 1, 2 and 3 days. The negative control was treated with cell lysate from MG1655 E.coli strain containing the empty vector. | ||
| - | |||
| - | |||
{{:Team:Imperial_College/Templates:footer}} | {{:Team:Imperial_College/Templates:footer}} | ||
Revision as of 20:51, 29 September 2013
Scanning Electron Microscopy
A way of showing that a degradation enzyme is functional is to incubate it with the polymer in question and scan the surface after some time. This is a widely used method for studying plastic degradation and we have taken advantage of it for characterising Proteinase K ([http://parts.igem.org/Part:BBa_K1149008 BBa_K1149008]) and observed how it "chews" on a PLA cup.
PLA can spontaneously hydrolyse in water. However, this only happens very slowly and at higher temperatures. Here is an SEM image from the literature (see reference below) where PLA was incubated for 9 and 20 days in deionized water at 58 °C. We are expecting our enzymes to have at least as visible effect in 3 or less days.
method
We cut small pieces of the cup and washed them with ethanol and deionised water in order to remove any potential contamination. Next, the substrates were incubated with cell lysate for 1, 2 and 3 days. The negative control was treated with cell lysate from MG1655 E.coli strain containing the empty vector.
