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From 2011.igem.org

8-29

The LB inoculated the day before yesterday had some precipitations in it after 40 hours. The experiment is a total failure because after PCR there were no segments at all!. We preserved and extracted plasmid from 18-3&11. But the results were bad.

We inoculated the DH10B，HB101 from the plated into LB.

Defect type BL21 grew up in the plate of MM+pro+VB1. 7 and 8 grew on the plate of MM+pro+vb1+leu but not on the plate of control plate. It is highly possible that these were Leu-defect bacteria.

The inoculated 5&4，leu,21&3,22&3 grew normally. And we selected the single colonies and inoculated them in the LB liquid media.

What’s more, we compounded TY media to cultivate rhizobium.

And, the first batch of 18-3&11-16 were transformed and inoculated on LB. The second batch had not grown.

8-30

The 5&4 and leu inoculated into LB liquid grew normally. 21&3 and 22&3 never grew.

The first batch that was inoculated into the LB liquid media did not grow. The second grew. But it turned out to be that we inoculated onto the wrong plate.

8-31

Today, we did enzyme digestion to 2 part (with psB1AK2 carrier) and K115001 part(with psB1AT3 carrier) and run electrophoresis together with psB1C3. We recycled the carrier part by cutting the gel.

And, we put some EP tube with unknown source in order and gave them two names: Those after enzyme digestion were named linkable and those that had already been transformed were given the name link-transformed.

9-1

Today we looked up to the ingredients of 0147TY medium and made it.

We then re-picked single colonies into LB liquid medium for shaking-incubation.

We re-ligated and transformed 18-3&11-16 and 18-3&11. Besides we found Spel enzyme (Takara) and competent cells have almost run out. So we decided to order some more.

9-2

As we reserved No.1 to No.4 of leu89 and contracted the plasmids, through enzyme digestion, the result still was not satisfactory with smear. After asking Haoqian Zhang we found the problem was on the enzyme digestion after PCR. At that time when we amplified leuB from BL21 by PCR, in order to get higher efficiency of enzyme digestion, we chose two inner sites Xbal and Spel. But we overlooked that these two enzymes are isocaudarners. Thus the fragments were messed, resulting in smear in electrophoresis. Consequently, we used E and P enzymes to digest the PCR product of leuB and cut the larger K381001 (psb1c3) gel to prepare for re-ligation.

There were colonies on 18-3&11-16 and 18-3&11 dishes coated yesterday. We picked single colonies for shaking incubation at noon.

We also poured TY medium to dishes.

Besides we have ordered five bags of Spel enzyme (Takara). As the deadline is approaching, we have to start the test of fluorescent protein expression. Today we transformed BBa_I751250 as the signal generator of Luxl_AHL. Then we transformed BBa_J04450 (placl+mRFP).

9-3

Firstly we ligated the vectors of leuB and psB1C3 and transformed it. Then we contracted the plasmids of 18-3&11-16 and 18-3&11.

Then we made IPTG solution. In the afternoon we put BBa_ I751250 (signal generator) in the shaking incubator. In the evening we added IPTG to induce the expression of Luxl. Besides we sterilized 0147TY solid medium, 0147TY liquid medium and LB solid medium. The we poured 0147TY solid medium to dishes with an addition of Str, LB solid medium with an addition of C and T antibiotics. Then we shaking-incubated 2-6&3 and No.2 part from the reservation tubes to Lb liquid medium for further use. In the evening we ligated 5&4 and 2-6&3 which digested by enzymes before.

9-4

Today we transformed 5-4&2-6-3, which were ligated yesterday; and contracted the plasmids of 2-6-3 and 2.

We filtered the I751250 bacilli with bacterial filtration membrane to get the AHL supernatant. In the afternoon we mixed Lux-AHL and LB liquid medium in the rate of 1:4 to culture 5&4 to get Las-AHL.

There were colonies cultured from LeuB dishes. We picked single colony to LB.

Besides, making deficient-type medium is kind of a big problem. Last time we used solid MM and SM for testing. The result showed basic confidence to use 1~5 of HB101 but the colonies were very small (compared to BL21). Hence we decided to set grades of VB1 in the medium and use liquid medium to test hopeful deficient-type colonies (No.1). The final mission is to transform the bought Pea Rhizobium.