Part 1

The standardization of four genes

We abstracted the target genes from the genome in E.coli K12 by PCR. However,there is one restriction enzyme site in ygfD that will has itself segmented without proper mutation.So we decided to obliterate the restriction site PstI by site-directed mutagenesis based on overlap extension PCR.After being sequenced,the four genes were linked to pmd18-T vector(Figure 1) for amplification and preservation.
In addition, we utilized an expression vector 2118CA (Figure 2) to make genes express in the DH5-α. By measuring the propionate production, we can know whether they truly work in the recombination vectors.
As iGEM officials require, the four genes are all standardized by adding EcoRI and PstI restriction enzyme sites at both terminates. They are all carried by psb1C3 vector (Figure 3).

Part 2

The construction of the biological oscillator

According to Jesse Stricker, they described an engineered genetic oscillator in Escherichia coli that is fast, robust and persistent. The oscillator consists two parts, activator with the reporter gene rfp, and repressor.
In our project, the activator and reporter modules are on a derivative of 2118CA whose original promoter is replaced by hybrid promoter (plac/ara-1) , which will be activated by AraC in the presence of arabinose and repressed by LacI in the absence of IPTG. While the repressor module is on a derivative of pET28-a (Figure 4).with its own LacI gene destroyed and a new LacI gene inserted. In accordance with the activator, the original promoter is also replaced by plac/ara-1.
We supposed that if the fluorescence protein changes periodically, then we can substitute our target genes for rfp.
Reference: Jesse Stricker, Scott Cookson, Matthew R. Bennett , William H. Mather, Lev S. Tsimring & Jeff Hasty. A fast, robust and tunable synthetic gene oscillator. Vol 456| 27 November 2008| doi:10.1038/nature07389

Part 3

The output evaluation of the propionate before and after the genes regulation

To prove the efficiency of gene regulation, the propionate generated in recombination E.coli BL21 strain as well as the wild type E.coli BL21 strain are measured by HPLC analysis. According to the growth curve of wild type BL21 under the 37℃, we found the optimum time to sample is 18h after the fermentation sets out. Cell growth was monitored as the optical density at a wavelength of 600 nm (OD600). At first, we did some experiment to explore better substrate for fermentation besides Luria- Bertani (LB) broth. We added glucose, succinate, vitamin B12 to LB broth respectively, and then measure the centrifugal supernatant after 18h. We use Kromasil reverse-phase C18 column (250X4.6mm, 5μm) under the conditions that are listed below:

Flow rate:

0.8ml/min

Mobile phase:

methanol : 0.005%KH2PO4(pH2.7)=0.1:0.9

Wavelength:

215nm