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From 2011.igem.org

9-5

In the morning we reserved the bacilli of leuB and contracted its plasmid. The next PCR and gel-electrophoresis showed nothing wrong.

In the afternoon we conducted double-enzyme digestion for 5 μl system of leuB and nothing was wrong. Then we added leuB into c-resistive LB liquid medium and took it back to Yushan campus.

Because of the failure on Pea Rhizobium yesterday, we continued on its activation today. We made new solution of T antibiotics concerning about its solubility in 50℃ hot water and folded it with foil. Then we added 54263 (loop I) to AT-resisting LB liquid medium

In the afternoon we made new leuB-deficient liquid medium (in gradient concentration of VB1) and added HB101 and BL121 into it.

9-6

We reserved colonies of 54263 coated yesterday, contracted the plasmid and conducted PCR and double-enzyme digestion. The resulted showed no problem on the part. But we doubted there were double plasmids.

We found the length of plasmid that contracted from K381001 was abnormal. It might be a mistake of the package received from MIT.

Yesterday we failed in the loop II experiment for it didn’t yield fluorescent protein testing bacteria. In the afternoon we incubated 18-3&11-16 in LB (AT+) medium without AHL.

There were colonies cultured from BL21 coated yesterday, while none was in HB101. So we kept incubating.

9-7

There was no colony cultured from 18-3&11-16 that were ligated yesterday. We found the reason was the wrong antibiotic (it should be K (- -)). So we changed the medium and re-cultured it with an addition of AHL.

We took the single colony of HB101 in deficient-type medium and determine its OD value. Then we selected the most concentrated as No. VB1② (compared to VB1). Then we coated the kidney bean and Alfalfa and incubated them in TY medium.

All the primers have arrived. We conducted PCR using these primers (12 pairs, 4 systems, 3 parts/groups).

The part of LeuB had been contracted so we can start to connect a loop to test leuB from today. Firstly we did the enzyme digestion for leuB, No3 and K115001 (T vector). We made three tubes of No.6 in TB liquid medium for shaking-incubation and determined the concentration of No.8, then condensed it.

9-8

Contract the plasmids form No.6 and check yesterday’s result of enzyme digestion by gel electrophoresis. The result showed no problem. Then we conducted large scale collection of T vector and determined its concentration. Besides, we did the ligation of leuB and No.3, and condensed No.8 part and did the ligation of it with plasmid of No.6 (concentration determined in advance) and leuB. The sites of digestion were: ⑥（E,S）；⑥（X,P）;leu(X,P);⑧（E,S）；In the meantime we use primers to conduct PCR of the part which were not successfully standardized yesterday.

In the afternoon we observed fluorescent protein in induced loop II.

Transformation of RFP-PSB1C3 was finished for further ligation.

We also reserved 18-3&11-16.

9-9

We tested the enzyme digestion result of No.8, No.6 and leuB by gel-electrophoresis and it showed no problem. So we continued to conduct the ligation of 8&6 and 6&leuB.

9-10

Today we conducted the transformation and coating of 8&6 and leuB. In the afternoon we inoculated leuB&3 from plat dishes to LB liquid medium for shaking incubation. In the test of loop by fluorescence, we cultured RFP-expressed bacteria in LB liquid medium for overnight and observed under fluorescent microscope. It showed a weak red color. We added IPTG into the bacilli and the red color became more obvious after 2 hours. And the control group did not show any changes, proving that RFP was normal. The over-all result of the fluorescence test of loops showed obvious fluorescence in loop I and weak fluorescence in loop II. Thus, we still doubted the problem was on No.16 part.

Besides we conducted PCR for sinR and raiR series. The result revealed that sinR pro(the promotor of sinR) and the amplified fragment of pro-RBS-raiR was on the right place.

9-11

In the afternoon we picked single colonies from dish-cultured 6&leuB which we coated yesterday to LB solution for shaking culture. There were no colonies on 8&6 dishes. We supposed that K-antibiotic was not valid so repeated the transformation and coated the product to new K+ dishes.

After enzyme digestion of RFPpart by E and P, we conducted the large scale of gel-collection to use its P5B1C3 vector to standardize the part. (the size of previous vector was wrong)

We used the four rested parts that needed to be standardized to continue PCR. raiR was successfully polymerized and was collected after enzyme digestion by E and P with the other seven parts that have been successfully polymerized.