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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.