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From 2011.igem.org

8-22

Today, we preserved and extracted plasmid from 18&3. At the same time, we found that there were colonies growing the other three parts (19-14&13；2-6&3和11&16). These meant that it was very possible that we succeed. We selected some colonies to inoculate and shake in the LB liquid media.

There were no colonies in the plate where phaseolus rhizobia were inoculated and where there is streptomycin. So today, we selected some bacteria from the preserving tube and the primary solid media and inoculated them into the TY liquid media to make sure that they did not get infected.

We did PCR again using Tan’s system. The results were quite good. The bands were right. After that, we recycle the products using PCR kits. In the evening, we did enzyme digestion to 18&3 plasmid. The next day, we would see the result by running electrophoresis.

8-23

In this morning, the linkage was verified. At the same time, we extracted the transformed 2-6&3；11&16；14-19&13 plasmids. In the afternoon, we did enzyme digestion. In the evening, we conducted single & double enzymes and them verify them. From the results of enzyme digestion, we could conform that the linkage was quite successful. Besides in the morning, we compounded the SMM media again. We added pro and Vb1 to them.

8-24

We run electrophoresis again to the three groups’ parts with the main aim to see the results of the double enzyme digestion. This time, the result was quite good and we can be sure that it was linked in the right way. But part 16 had some problems. The part showed in the electrophoresis and the size of plasmid were not consistent to the official wesite. So we suspected that something is wrong with the part given. We decided to test them according to their function (that they can express Fluorescent protein).

It was a pity that there were new problems in the SMM media compounded yesterday. It was that we did not use the control group of MM with Vb1 or pro. In the way, it was possible that if the bacteria were only Vb1 and Pro defect but not Lew defect, they cannot grow in the SMM media. And they would be mistakenly seen as the Leu defect. So today, we compounded two media and they are (MM+pro+Vb1) and (MM+pro+Vb1+leu).

8-25

Today we inoculate HB101 and BL21 into the solid medium (MM+VB1+Pro and MM+VB1+Pro+Leu). In the afternoon we culture the Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 on TY solid medium at 30℃.

In the evening we do the transformation of leu(13/45/67/89) and part 18-3&11-16 at 11:00,which finishes at 4:00 in the morning.

8-26

We make LB liquid medium. We do the enzyme digest of part 22;21;3 to prepare for the ligation using the traditional method.

8-27

In the morning, we transformed and inoculated 18-3&11 using 3A method. The 18-3&11-16 transformed yesterday did not show up. We transferred 5&4 and leu into LB liquid medium. We recycled 22, 21, 3 from the gel. And decided to link 21&3, 22&3 using the traditional method.

The two plates used to verify defect types did not grow.

In the afternoon, we inoculated K115001 in pSB1AT3 into LB liquid media and shook them.

In the evening, we lined 21&3, 22&3.

8-28

Today, we found that the solid media with antibiotic was not good at all. So we compounded them again, and transformed 5&4 again, leu.

In the morning, we transformed 21& and 22&3 and cultivated them on plates. We inoculated the colonies on 18-3&11 plate into the LB liquid media.

There was no precipitation in the LB media at noon. There are some in the afternoon.

In the afternoon, we inoculated the bacteria on the defect type plate again.

In the evening, we transformed 18-3&11-16 again and spreading them.