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Team:KAIST-Korea/Notebook/Week2
From 2011.igem.org
Week 2
6/11~12 Saturday~Sunday
Seoneung, Jeonghyun, Joonhyuk
@ KIB
By yesterday, we had two problems, one in the gel electrophoresis and the other in the competent cells, and decided to examine whether the mini-prep or the gel didn’t work by another mini-prep and gel electrophoresis and to make other competent cells, respectively, in order to solve each problem.
During this weekend, we performed re-experiments in order to solve the problems caused in last week.
Tomorrow, we’re going to discuss on the problems that have not solved yet and perform experiments again so that we will be able to go forward.
6/13 Monday
All wet lab members
1 p.m. - 9 p.m. @ KIB
During last weekend, we conducted re-experiments in order to solve the problems caused in the gel electrophoresis experiment – no band corresponding to either the plasmid vectors or the biobrick inserts.
Today, we’ve done two things. First, after a brief discussion, we continued conducting re-experiments in order to solve the ‘no band’ problems by examining whether the used gel, the way of gel electrophoresis (two cut by the restriction enzymes), the used restriction enzymes, the mini-prep procedure, the competent cells, or the media plates didn’t work properly. We could reach the conclusion that the gel and the restriction enzymes were the reason for the problems and 3 biobricks – J61047, J37033, E0420 – needed transforming again due to their no formation of the corresponding bands in the electrophoresis. In addition, we tried making other competent cells on our own. Second, we had the lab meeting with dry lab members. Dry lab said they had got the problem in using the program Tintkercell and decided to employ another program, MATLAB. In addition, they explained how they did modeling, i.e., how to calculate the relationship between the amounts of IPTG that is put and the light intensity that E. coli emits.
Tomorrow, we’re going to check if the competent cells grow properly and if not, we’re going to make competent cells again.
6/14 Tuesday
All dry lab members
@ KIB
In searching for efficient ways to model our genetic circuit in silico, we came across Tinkercell. The program allows the user to click-and-drag components of the genetic circuit, such as promoter, ribosomal binding site, and terminator, to graphically design a genetic circuit. Although this program was great for synthetic biology, we abandoned using it and instead used Matlab because doing so provided the optimum customization that we needed.
All wet lab members
@ KIB
By yesterday, we had been concentrating on solving the problems that were spotted in the gel electrophoresis and making new competent cells that we would use during the whole project.
Today, we’ve done six things. First, we ordered what we need such as the restriction enzymes, the ligases, and the antibiotics. Second, we continued making the competent cells. Third, we did mini-prep again on the biobricks that either didn’t show the bands in the electrophoresis or we hadn’t extracted their plasmids. Fourth, we started making another kind of competent cells called Top10 due to the low efficiency of the XL-blue cells. Fifth, we briefly cleaned and arranged the lab. Finally, we had a brief discussion on how to construct the biobricks.
Tomorrow, we’re going to continue making the competent cells. We may employ another protocol for making competent cells since the XL-blue cells, which we are using now, don’t seem good in their conditions.
6/15 Wednesday
All wet lab members
@ KIB
So far, we had been concentrating on making competent cells that will show high efficiency as well as transforming the biobricks that need transforming again.
Today, we’ve done four things. First, we continued making the Top10 competent cells, after which we checked the efficiency of the competent cells. Second, we discussed on whether we would use the linearized plasmid from the distributed biobrick kit. Because it had been kept for a pretty long time, we decided not to use it. Third, we continued conducting mini-prep with the 7 biobricks that needed transforming again to check if the transformation had been achieved successfully.
Tomorrow, we are going to begin constructing the biobrick assemblies by putting together the biobricks through the 3A assembly method using the restriction enzymes and the ligases.
6/17 Friday
All wet lab members except Jooyoung
@ KIB
Today, we have done three things.
First, we continued trying to guarantee the mini-prep samples of all of the biobricks. We conducted mini-prep with the E0420 that seemed to be one of the problematic biobricks. And we conducted the gel electrophoresis with the 8 samples (6 problematic plasmids’ samples + E0420 + 1 more Q04121). By the way, the J37033 sample didn’t form any bands in the electrophoresis and we conducted the gel electrophoresis with the original biobrick sample of the J37033 as well as re-transformed it. In addition, Q04121 and E0420 didn’t show the proper bands, which made us treat them with the restriction enzymes for overnight this time (in the previous case, we conducted one-hour treatment on E0420).
Second, we continued the experiments to guarantee the three construct plasmids’ mini-prep samples. We checked their colonies and amplified them by seeding into the liquid LB media.
Finally, we got another protocol for the restriction enzyme treatment as well as PstI, a required restriction enzyme in the project.
In conclusion, we continued performing the experiments on the biobricks and the construct plasmids, and prepared materials and protocols in order to proceed to the biobrick assembly using 3A assembly.
6/18 Saturday
Seoneung, Sohyun, Youngjun
@ KIB
Today, we have done two things.
First, we checked the band formation of the three problematic biobricks – E0420, Q04121, and J37033 – by the gel electrophoresis. Due to the failure of finding the corresponding bands to the vectors and inserts of the biobricks, we decided to start from the transformation of the three biobricks and this time, however, pick up the three colonies in amplification.
Second, we continued preparing the mini-prep samples of the three construct plasmids by mini-preping and cutting the parts of the samples with the restriction enzymes.
In conclusion, we continued making an effort to guarantee the proper mini-prep samples of all of the required biobricks as well as the three construct plasmids required for the 3A assembly.
