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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.