Monday, July 23 Today we sonicated three cultures of E. coli - one with our Z construct, one with Z, C and inducers, and one with Z, C, but no inducer. Then, we extracted the soluble proteins from those cultures and ran them on a gel. We also ran a PCR of a series of ligations. The color constructs were ligated with promoters into chloramphenicol-resistant plasmid Psb1C3. [EXPAND UPON THIS] We extracted the proteins from our cultures of E. coli (with our Z construct, with Z and C construct and non-induced, and with Z and C construct plus inducers) and took spectrophotometric readings
Today, we ordered multiple primers that we will need in the future.
We also performed a ligation of RFP construct with PSL2131, and then we plated the results to check tomorrow.
Wednesday, July 25
We checked the ligation plates of RFP and PSL2131 and see red colonies, indicating that the ligation was successful. After the colonies have a chance to grow a little more, we will start cultures and miniprep them.
We have discovered that we need a fusion protein in order to deal with insoluble protein products that form inclusion bodies, preventing the effective transcription of our gene. Thus, we obtained the pET-42a-a(+) vector, which has a GST tag, from the lab of James Havranek.
We also ran a four-hour experiment to test the effects of our inducers (IPTG and arabinose) on the promoter for the zeaxanthin construct in E. coli. To do this, we varied the concentrations of both of the inducers, with 0 or 1 mM IPTG and 0.02%, 0.2%, and 2% arabinose. We collected OD450 and OD600 measurements every hour. We will take one more reading tomorrow morning and then analyze the data obtained from the experiment. Thus, we have now successfully characterized an existing part! Please see our characterization page for further details.
Finally, we digested Psb1C3 with RFP with E and P.
Thursday, July 26
After taking the final OD readings of the E. coli cultures today, we extracted the carotenoids from the tubes and took spectrophotometric readings on them as well.
We also started a culture of E. coli transformed with the Z construct to be run on an SDS-PAGE gel. Around 3PM, we checked the OD600, found it had reached the peak growing stage (determined by an OD600 of .4), added the optimal amount of inducer determined by characterization on Wednesday (1 mM IPTG and .2% arabinose) and let the culture grow. Tomorrow, we will sonicate and run on the gel.
We miniprepped the ligation of RFP and PSL2131, digested it and ran it all on a gel to check that the ligation occurred.
Finally, we performed a digestion of Z construct and CS42S and ran it on a gel.
Friday, July 27
This morning, at 9AM, we retrieved the Z-transformed E. coli culture from the previous day, and, after treating the cells as per the cell lysis protocol, we ran the samples on a gel. Once the gel had run completely, we stained and left it to destain overnight.
Miniprep + gel of stuff
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