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From 2011.igem.org

9-12

Since there was only one of the three tubes of 6&leuB turned turbid, we took some bacteria from it for reservation and examination. The rate of A260/A280 was not normal and the result of gel electrophoresis was not satisfactory, either.

There were still no colonies on plat solid medium so we repeated the ligation today.

We conducted enzyme digestion of No.21 and No.22 for future ligation with the digested product of No.3.

In the afternoon we tested loop ligation by signal molecular. For loop II, we used lux&las and lux(control group) as inducer. For loop III, we used lux&las&cin and lux&las(control group). Tomorrow we will get the result by observing under fluorescent microscope.

9-13

Today we repeated PCR process using yesterday’s templates (20 samples, 10 pairs of part). But the gel electrophoresis all showed smears.

We selected 21&3, 22&3, 8&6 to conduct the transformation, coat-cultured the product, continued the ligation with PSB1C3 vector and large scale of gel recollection.

The result of screening of the signal-molecular-induced product showed that the loop of lux&las in loop II appeared weak fluorescence. The lux-reduced loop II did not show fluorescence (control group). We supposed that there was something wrong with No.16 fluorescent protein and decided to re-build loop II.

Since the bacilli did not turn turbid, we re-inoculated the single colony from dish-cultured loop III bacteria to LB medium.

9-14

After 20 hours, the parts—21&3,22&3,8&6 haven’t grown out, but test tube with circuit-III’s have been turbid.

Ligate the parts—sinR,sinI and pro(sin) with the carrier C to be standardized.

By detecting, the part pro-RBS-sinR’s electrophoretic band is too shallow, and the part pro-RBS-raiI, pro-RBS-raiR and pro-RBS-sinI’s bands are all blended, and the sinR has less blended bands. We retrieve the sinR,sinRpro,raiI-pro,sinI-pro and lay down the others.

The part NO.16 in Circuit-II is proved that its length doesn’t accord with the standerd.We suspect it’s the part’s questions. Then we decided to ligate the NO.16 with small parts composed the NO.16. Today C0076 and E0020 have been transformed.

9-15

The standardized five parts are transformed at 10 a.m. and coated on square plate at 2 p.m.

6&leu’s first test tube is proved to be ligated correctly.

21&3 and 22&3’s bacteria colony haven’t appeared on the plates.So we have to isolate the plasmids again, and put them in LB culture medium.

8&6 are enlargement cultured again. 6&len are double-enzyme cutd in the evening to be ligated in the traditional method and 3A method.

We pick ou the bacteria colony of E0020 and C0076, and put them in LB culture medium with shake cultivation at 37℃.

9-17

After detecting Leu,we find the plasmid in no.2 and no.3 tude still can be used. Take some bacteria in LB culture medium.

9-18

Detect leu and 6 which have been cut and then ligate them in 3A method.

Cut 6 for the second time at 5:40 p.m. to ligate with leu in 3A method.

Keep on standardize parts including raiR1,raiR2,raiR3,raiI,raiI2,sinR1,sinR2,sinR3,sinI1,sinI2,sinI3,psinI1,psinI2.