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From 2011.igem.org

9-26

Extract the plasmids of 14-19 and RFP and detect their concentrations.

Pick out the bacteria colony of J13002 into LB medium.

The plates which are used for standerdizing 18-3-11,14-19,5-4,rhiABCpro,13-6-leu and 18-3-11-6-leuB have already bacteria. And we select some into LB.

Double-enzyme cut RFP, rhiR,14-19-13 and LeuB with EcoRI and PstI. We recover them with gel extraction mini kit after electrophoresis.

Standerdize the part—rhiI. Transform C0076&6、E0020&6、5-4&6-leuB and coat them on square plates. Then ligate rhiR&RFP, leuB&carrier C, 14-19-13&carrier C.

9-27

Preservate the strains of J13002 and extract the plasmids. Circuit-I’s standerdization failed. We go on cleavaging, ligating and transforming. At the same time we extract the plasmids of 18-3-11、14-19、5&4、rhiABCpro、13&6-leuB、18-3-11&6-leuB. But the plasmids of 14-19 go red because of ligating themselves. We have to lay it down.

We put one preservation tube of every strain into -80℃.

Four parts transformed yesterday failed.(so many failures…oh no>..<) Transform the standardized parts of rhiI、14-19-13、leuB、rhiR. Verify the standardized parts by double-enzyme cleavaging.

Make some solid medium. The resistance are K and T.

In the evening, we cut RFP (to be standardized),14-19(to be standardized), J13002(to be verified) and 6-leuB(to be 6-leuB).

9-28

At 12:45 a.m, put the standardized parts—rhi,-4&6-leu,6&C0076 in LB to be shaking cultured. In the morning we make up 1000ml TAE. Then double-enzyme cut the standardized parts to be verified after electrophoresis. We find the length of 5-4 is questionable. So we decide to verify it again by shaking culture three tubes of bacteria. The length of 6-leu-13 is also questional. It seems that it haven’t been ligated with 13. We have to ligate them again. 18-3-11-6-leuB is ligated successfully. And 18-3-11 and rhiABCpro’s standardizations are successful.

Pick out the bacteria colony of 6-E0020 into LB. In the afternoon, coat 5-4-6-leu and 6-E0020 on the square plates. We ligate 6 with C0076 in traditional methods.

Extract the plasmids of 5-4-6-leuB and cut it.

9-29

Before dawn, we transform six tubes. These are: re-ligated leu-6-13 and standardized rhi,leuB,14-19-13,14-19,circuit-I.

At 1 p.m, we extract the plasmids of 6-E0020. Then verify the enzyme cut 5-4-6-leuB. The results are the plasmids’ length are both right. But the first tube’s cut plasmid’s length are wrong. So we use the second tube’s plasmids.

In the daytime, we enzyme cut 6-E0020,no.3 and 2-6-3. The electrophoretic band of 6-E0020 is right! It can be ligated with no.3’s cut products. Ligate 2-6-3 with 5-4-6-leuB.

9-30

At 11 a.m, transform 5-4-6-leu-2-6-3 and 6-E0020-3. At 3 p.m ,it’s time to coat plates’ culture. Pick out the colonies of 6-C0076 and standerdized leuB into LB liquid.

Standerdized partrhiR, 14-19 and circuit-I haven’t grew out. No.3’s electrophoretic bands is questional. So we enzyme cut them and verify by electrophoresis again. It’s correct. In the evening, we activate circuit-I’s strain and culture them in LB medium. Begin to the work of wiki and kinds of things about going to Hong Kong .

10-1

Today is National Day! Such a great day~~-

We have many tasks of wiki. We all have our own tasks and prepare to perfect our wiki.

Verify 6-C0076 by electrophoresis and find they haven’t been ligated. So we cutd them and 6-E0020&3 again. Then pick out colonies of HB101 and I751250 into LB to make the defective competent cells. Extract the plasmids of circuit-I. In the evening, ligate 6&C0076 and 6-E0020&3.

Standerdizing parts is going on. We prepare to send the strains.

10-2

We do a precise estimation and find circuit-II and III can’t be ligated on time. So we decide to put the point to circuit-I’s ligation and prepare to verify it. We make the defective competent cells and extract the signal molecule. We transform circuit-I into the defective cells. Wait for the results.

Wiki is extremely intense. Many pictures need to be made and so many things need to be translated in English. DNA submission is on its way, but we encountered frustrating affairs, like the non-working day’s all kinds of inconvenience, and problems of sending liquid items when at Chinese customs. We have to use the filter paper transferring, only to detect that we don’t have gresol red indicator. All the unlucky things…… Pray for that our shipment will be in time.