Team:Athens/Methods
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Methods
ALT SEL - X
ΑLT SEL - X is simply the name we use for our general experimental process; but if the words allow for such an elegant abbreviation, it would be a pity not to use it! The letters stand for:
Amplify - Ligate - Transform - Stir - Evolve - Lyse
Amplify: The first step of the process is to select the initial aptamer sequence you wish to subject to directed evolution. Make sure to “amplify” your sequence, in other words, obtain sufficient quantities for the next steps.
Ligate: once you have your sequence, it is time to insert it in the Aptamer Binding Cassette (ABC)! We have designed our system so that the aptamer sequence site is interchangeable, so all you have to do is digest with SexAI and KpnI and ligate!
Transform: Transform your plasmid into E. coli cells. As the plasmid is quite big in size, we recommend you use highly competent cells. Find the transformation protocol, and many more, below.
Stir: Stir, shake, incubate your transformed bacteria! You can also build your own DIY morbidostat to support a constant bacterial culture with constant evolution pressures!
Evolve: Let the system work! The cells will start expressing the mutator module which will act on the aptamer sequence!
Lyse: After a few days, the cells that will have survived will be the ones expressing evolved aptamers with higher affinity for a specific target molecule. Lyse the cells (a.k.a. miniprep ;) ) to obtain the plasmid and then isolate and sequence the new aptamer!
Find below all the protocols we used in the lab! It is, also, possible to download them as .pdf files!
Methods