Team:KAIST-Korea/Notebook/Week4

Week 4

6/27 Monday

Joonhyuk, Seoneung, Sohyun, Jeonghyun, Youngjun

@ KIB

We had difficulty assembling the biobricks last week - no band results on the gel electrophoresis. We're checking our protocols of restriction enzyme treatment and ligation. In order to check which procedure was improperly done, we've done three things today.

First, we tried to extract the construction plasmids C3 by the gel extraction. By the way we failed to observe the bands corresponding to the vector C3 that don't contain the inserts although there was the one-cut band of the construction plasmid. We thought there might be a problem in the restriction enzyme protocol.

For the next step, we tried to check if the restriction enzymes have properly worked on the biobrick plasmids (not the construction plasmids). At first, we conducted the gel electrophoresis with the biobricks of the pairs #1 to #12 (arbitrary names made by us) using Xba I and Pst I. By the way the results didn't show any bands corresponding to the vectors and the assembled inserts. We concluded that there was problem either in the protocol of the restriction enzyme treatment or the restriction enzymes themselves.

Fianlly, we decided to check the process of the restriction enzyme treatment and started the experiment by treating the biobricks of the pairs #1 to #12 as well as the construction plasmids C3 with Xba I and Pst I (overnight this time, not 2 hours). We expected to discover where the problem lies between the protocol and the enzymes by this experiment.

In summary, trying to solve the problems in assembling the biobricks, we discovered there was the problem in the restriction enzyme treatment and started checking where the problem lies - either on protocol, enzyme, or both of them. Tomorrow, we're going to check if the proper bands appear in the gel electrophoresis with the biobricks of the pairs #1 to #12 and do something additional according to the results obtained.

6/28 Tuesday

Jeonghyun, Sohyun, Youngjun

@ KIB

We discovered there was the problem in the restriction enzyme treatment (RET). Today, we continued trying to find where the problem lies by conducting the similar experiments with those of yesterday - two kinds of gel electrophoresis (GEs).

First, in order to check if the REs had properly worked, we conducted the GE with the biobricks #1 to #12 and the construction plasmid C3. The results showed only one-cut bands and gave a conclusion that one of the REs, Xba I and Pst I, broke down.

So, in order to check which of the REs is the problem, we conducted the GE with the biobricks of the pairs #1 to #12 except E0040. Most of the bands showed the proper sizes, which means good results and made us confused. We made a conclusion that the amount of the restriction enzymes we used on the two-cut experiments was too low to cut both points on the biobrick plasmids.

In summary, trying to solve the problem of the RET procedure, we discovered one of the REs, Xba I and Pst I, caused the problem, investigated which of them didn't work properly, obtained the results that were seemingly weird, and finally, guessed the amount of the RE was too low. Tomorrow, we're going to go to MT and continue the experiment three days from today.

6/29~30 Wednesday~Thursday

All members

Group outing

We went to a guest house near a gorge in Chungbuk Province for a two day program designed to help us get closer to each other. We played by the flowing river, told stories, ate barbecue, and drank some alcohol! ;)

7/1~7/2 Friday~Saturday

@ KIB

We have difficulty cutting the biobrick plasmids using the restriction enzymes.

So, for two days, we’ve checked the protocol of the restriction enzyme treatment and tried improved the protocol by modifying it according to the experiments for check.

We’re going to continue checking the restriction enzyme treatment step since we’ve not been satisfied with the results in the experiments for two days.