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From 2011.igem.org

8-15

Today, we used the old method to do single & double enzyme digestion to 18&3；2-6&3；11&16. However, in the evening, the electrophoresis result was not good at all. Besides, the new 3A method PSB1AC3 plasmid backbone（K131017）. At 15.30, plate was processed and would be put the liquid media the next day. What’s more, we find plasmid PSB1C3 in plate 4’s 1A. 1E, 1L. These were used to standardize the part. In order not to waste the sensitive cell, we decided to transform them together with other part.

8-17

At 8:30, we preserved the K131017. After that we extracted ten tubes of plasmid, the concentrations of which are high, at over 100ng/ul. In the afternoon, we did enzyme digestion to these plasmids. After that we run small electrophoresis to verify them anddig big one to recyle the 3055 band. We prepared to link the 5&4 from previous experiments and the recylcled K131017.

And we were also sifting the defect bacteria. Today, we did streak cultivation to LeuB6. And we also compounded the MM and SMM and the TY media used to cultivate phaseolus rhizobia. MM and SMM were vey special, some part of which needed to be sterilized separately under 55oC. Some precipitation were produced after mixture.

8-18

In the morning, we did linkage of 5&4. In the afternoon, we transformed them and the part in P4-1A.

At the same time, we put the LeuB6-cultivated colony into the other two LB media. The two media are divided into 11 equal plate. After the bacteria grow up, we put them into different MM and SMM.

In the afternoon, we tried to run PCR and verify them using gel electrophoresis. The results were no good.

In the evening, we put the three kinds of phaseolus rhizobia (meliloti 1021 SmR, CFN42 SmR and wild CFN42). And we put them in the oven.

8-19

MM and SMM were to soft to be eligible. And today, we are going to recompound them. We found that we had failed to compound the solution to right concentration. It was 5*. We guessed that this is the reason that precipitation was generated last time we compound the media.

5&4 plate had a lot of colonies. But we are not sure this was the right linkage. In the afternoon at 16:00, the LB liquid media were shaken. The next day, the result would be out. Today we conducted enzyme digestion to 18, 3, 11, 16, 2-6, 14-19. And the next day, we would do linkage together with other linkage. What’s more, we compounded some PY liquid media. The next day, we will be able to inoculate them and have the shaken.

8-20

In the morning, we extracted plasmids from 5&4 and linked the 4 groups from yesterday. In the afternoon, we did enzyme digestion to verify them, but it turned out to be a fiasco. So today, after we transformed them, we added antibiotic in them and expected there would be good results.

In the morning, we inoculated the LB defect type into the MM and SMM. They were still quite soft, but could be used more or less.

There are three plates on which phaseolus rhizobia were inoculated. Only one of them turned out to be successful. The others were total failure and needed to be done all over again.

8-21

In the morning, we run electrophoresis to the transformed plasmid 5&4, digested plasmid and plasmid from 5 to verify them again. We were sure that these combined plasmid were 5 linked to itself.

The 4 groups of plasmid transformed yesterday were quite frustrating. By 2:00 in the afternoon, only 18&3 have some colonies on them, others did not. We guessed that in the media with chloromycetin, it takes a lot of time for the bacteria to grow up.

And ,we preserved K381001 and extracted plasmid form them. And we put SmR in the TY liquid media and inoculated phaseolus rhizobia. We put the bacteria BL21 which is for PCR onto the LB plate.

Today, the inoculated defect bacteria did not grow up in SMM media. After we sought help from Tan, we, again, found we were quite wrong when we compounded the media. The reason is that the Leu gene was knocked out at the same time when other genes were knocked out. For example, in the HB101 we used, the genes used to express the thiamine and Pro were also knocked out so that we need to add the two things in the SMM media to let them grow.