·Materials

·Methods

Step 1 : To find the optimal temperature for araC amplification, we set a gradient in temperature . Then we amplified araC sequence by PCR in 58.5℃. The sequence was stored in -20℃. The PCR conditions were listed as table 1-3.

Step 2 : Digest 2118CA vector(fig1-1) to dismiss its original promoter and 2512EA vector(fig1-2) to donate a hybrid promoter with EcoR I and SpeI. Retrieve and purify the target genes with kits produced by. fig1-3 was the gel image of the skeleton and hybrid promoter. Afterwards,2118CA’s skeleton and hybrid promoter were linked together to recombine a new vector：2118CA+hp. The conditions for gene digest and conjunction were listed in the table 1-4,1-5.

Step 3 : To verify the linking reaction was correct, we had the conjunct gene digested with EcoR I and SpeI, following that we loaded the 2118CA+hp plasmid in the agarose gel electrophoresis. The final image was displaced in fig1-4.

Step 4 : Dispose previous araC gene sequence with XbaI and SpeI to expose the restriction enzyme site at both terminals. Digest 2118CA+hp plasmid with Spel to create an incision for inserting araC fragment. After verifying two fragments using agarose gel electrophoresis(fig1-5), they were linked together creating the active feedback part.

Step 5 : The novel plasmid was transferred into BL21strain, which later was grew in the Amp+ LB broth, 37℃ for one night. To ensure the conjunctions were correct, we confirmed the vectors by colonial PCR and gel electrophoresis, following the digestion of SpeI and PstI to see the possibilities of reverse connection. It was proven that one out of 10 mono-clones we picked was in the right in the marker ladder but reverse connected in the plasmid. So we picked mono-colonies and testified again and again to harvest correct plasmid in considerable concentration. (See fig1-6) The conditions for gene conjunction/ colonial PCR / dual-enzyme digestion reaction were listed in the table 1-6,1-7,1-8.

Step 6 : After successfully constructing the active part of the dual-feedback oscillator, we engaged to build the repressor part, pET28 vector with a replaced lacI structural gene and a hybrid promoter. As the sequences of original lacI in pET28 have the restriction recognizing sites namely Mul I and BssH II, we chose the way of digestion by these two enzymes to dismiss the original lacI. Then we did the next gene conjunction to introduce hybrid promoter and a new lacI gene on condition that the previous result was validated. The general operations were similar, so there should be a reasonable ellipsis.

Step 7 : Even we had our oscillator constructed, to coherent with the modeling mates’ opinion, we added LAA tags to the end of araC and rfp reporter gene to accelerate degradation. To increase the transformation percent, we cut the sequence of hybrid-araC-LAA-rfp-LAA from 2118CA and pasted on pET28 vector.