Our project is divided into three parts:

Part 1 : The construction of the biological oscillator

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 ...Read more

Part 2 : The output evaluation of the propionate before and after the genes regulation

Step 1 : PCR amplification with the primer(rbs-f/std-r) set as the template (E.coli K12 genome) will produce four fragments namely ygfG(786bp), ygfH(1479bp), ygfD(996bp) and Sbm(2145bp) with RBS and standard suffix added. And to follow we used PCR production as template and primer pair (std-f/std-r) to add standard prefix. The conditions of the reaction were listed in table 2-3 and fig 2-1 displayed the result testified by 1% agarose gel electrophoresis... Read more

Part 3 : The standardization of four genes

Step 1 : PCR amplification with the four pairs of primer set as the template (E.coli genome) will produce four fragments namely ygfG(785bp), ygfH(1478bp), ygfD(995bp) and sbm(2144bp). The conditions of the reaction were listed in table 3 and figure 3-1 displayed the result testified by 1% agarose gel electrophoresis. Step 2 : It is necessary to add PstI and standard restriction enzyme sites to both terminals of each gene. However, ygfD has the sequence that PstI recognize and function. So we obliterated the restriction site by site-directed mutagenesis based on overlap extension PCR... Read more

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