IGEM:Brown/2007/Tri-Stable/2007-7-27

Protein Tags
We discovered that all of the FP that we had planned on using were tagged with LVA. I guess this wasnt so much of a discovery as we disigned them to degrade fast in order to minimize the effect already existing proteins. What we didnt realize was that LVA tags degrade the proteins just about as fast as they are made (the degradation half life and folding are about 50 mins), confirmed by Jason Kelly and an article Norris read on tagging GFP. This renders the FP undetectable in experiment. We instead are using ASV tagged FP for the test (moderate degradation) and AAV tagged FPs for our switch (slow degradation). This should enable us to have a clear readout for our test and switch. This, however has made us redisign our ligations for the 3rd, hopefully last time. We have decided to only use ERBS (1.0) and SRBS (.6) in our preliminary switch until our tests show otherwise. This is due to yet more somber news.

All of our repressors are LVA tagged. And there arent any non tagged or ASV tagged repressors in the registry as of yet. This has us quite concerned about how strong of a repression we will be able to obtain out of our system. According to our model, the stronger the repression the more potential stability the switch could obtain. BioBricking non tagged repressors may be on the menu for us in the near future.

Critical Mini Prep Spin RPM
We tried mini prepping DNA using a centrifuge that only goes up to 10,000RPM, thinking that the extra 3,000 rpm of 13,000rpm wasnt really necessary. After two rounds of low yield, we had to conclude that the extra 3,000rpm was mandatory. My thinking was at lower speeds, there isnt a great enough force exerted on the DNA in the spin column for the final step, thus, it wasnt able to be spun through the filter.

Mini Prep Control
We tried running a control for a mini prep, mini prepping cells with no amp resistance (ie our plasmids) to see how much backround genomic DNA we were getting in our samples. After two tries and similar cell concetration n the control as the normal miniprep, we had the same if not a higher concentration in our control as our normal minipreps. We think that this concentration is not due to genomic DNA but rather to plasmids already in our cells (XL1Blue). We will run a gel on the control mini prep to see if that is the case.