Monday, July 9
Today, we maxi-prepped our construct CS42S, digested both CS42S and our plasmid PSL2131, and ran them both on gels. We made one-well gels in order to allow us to gel purify the constructs and recover DNA.
Then we gel purified the construct and plasmid and nanodropped them.
We went to the YLC for our initial visit today. After giving our talk, we ran the experiment and brought the plates back to incubate them until Wednesday, when we will return to conclude our experience with the YLC students. Tuesday, July 10 We learned that the biobrick protocol for ligation was not optimal for our conditions. Thus, using a different protocol (General Ligation Protocol), we performed a ligation of our construct CS42S and plasmid PSL2131. Then, we transformed some E. coli with the results of our ligation and put the plates in the incubator. In addition, as an extra check, we ran a gel of our ligation results to see if the procedure had worked. The gel reveals that we have several different ligations going on, including the ligation that we expect to see, at around 9 kb. In the gel below, each well has one tube of our ligation results, and two ladders are placed on either side of the trials.
We finally proceeded to transform E. coli with our ligations and put the plates in the incubator to check the next day. Wednesday, July 11
After two days now, we returned to the YLC with the students' plates to show them their results and conclude with a talk on our project. Pictures of the children's plates can be seen on our social media page.
We examined the transformed plates, picked a colony from our one successful plate, and grew it up in LB. At the end of the day, we miniprepped the result and nanodropped it. Tomorrow, we will run a gel on it and digest it to see if the colony had the correct ligation.
Thursday, July 12
The digestion of the miniprep from Wednesday, when run on a gel, revealed that the colony was not the ligation that we wanted.
Thus, we digested our construct CS42S and plasmid PSL2131 anew, gel purified the two, and performed a second ligation. Again, we ran a gel to check that the ligation was working. The three numbers correspond to the three different tubes we prepared when running the ligation. Tubes 1 and 3 included the heat inactivation step whereas tube 2 did not, in order to test whether heat inactivation is a necessary component of ligations.
Miniprep new parts + glycerol stock of new parts Started a maxiprep culture of zeaxanthin E. coli construct
Friday, July 13
Clone zeaxanthin and CS42S constructs into GC5 and Bl21 cells. Four clonings - Bl21: CS42S and CS42S + Zeaxanthin, GC5:CS42S and CS42S + Zeaxanthin. Then, these clonings were plated out and grown up in the incubator.
After digesting PsbA2 (promoter) with Z, U and C, we ligated the results with plasmid PSL2131. We performed three triple ligations - each one consisted of a promoter, plasmid, and then either Z, U or C, following the instructions detailed in the biobrick assembly protocol.
We ran a maxiprep to obtain DNA from our zeaxanthin maxiprep culture from yesterday and then nanodropped the result to obtain the concentration of DNA.
We have also started Maxiprep culture for psbA2, in order to have sufficient promoter DNA for future ligations.
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