·Materials

Table 1-1 Bacterial strains and plasmids

Strains and vectors	Relevant genotype and characteristics	Originate
E.coli DH5 α	strains	conserved in the lab
pET-28a(+)	vectors	conserved in the lab
pSB1C3 vectors	vectors	conserved in the lab

·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.

Table 1-3 Gradient PCR to harvest araC

System components(30μl)	Volume
ddH2O	17.1
PrimerStar Buffer	6
dNTP	3
PR	1.5
PF	1.5
Template	0.6
TPrimerStar	0.3

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.

Table 1-4 Conditions for digestion

System components(50μl)	Conditions
10×H Buffer 5μl	37℃ 1h
EocRI 2.5μl	37℃ 1h
PstI 2.5μl 10μl	37℃ 1h
ddH2O up to 50μl	37℃ 1h

Table 1-5 Conditions for gene conjunction

Item	System components(50μl)
Solution I	5ul
DNA	4.5ul
pMD18T	0.5ul
Conditions	16℃ 2h

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, 370C 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.

Table 1-6 Conditions for gene conjunction

Item	System components(50μl)	Conditions
1	Solution I 5μl	16℃ 2h
2	DNA 4.5μl	16℃ 2h
3	pMD18T 0.5μl	16℃ 2h

Table 1-7 Conditions for colonial PCR
(*Item 2 to item 4 for 34 cycles)

Item	System components(50μl)	Conditions
1	2×EX Tag Mix 5μl	94℃5min
2	M13-47(10μmol/L)0.5μl	94℃30sec
3	M13-48(10μmol/L)0.5μl	58℃30sec
4	ddH2O up to 10μl	72℃(1min for ygfG, ygfD 1min30sec for ygfH 2min for Sbm) 10min

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.