Overview

According to Jesse Stricker, they described an engineered genetic oscillator in Escherichia coli that is fast, robust and persistent. The oscillator consists of 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 1).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

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 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

Figure 2 : The structure of pMD18-t vector

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 2) for amplification and preservation.
In addition, we utilized an expression vector 2118CA (Figure 3) to make genes express in the DH-5α. 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 4).

Results

1 .Biobricks (we submit / Registry ) table describe

Table 3-11: Information of four standard biobricks

Standard biobricks	description
pSB1C3-ygfD(EocRI PstI）	According to Toomas Haller, the enzyme encoded by the second gene, ygfD, contains a consensus binding sequence for ATP. They thought it might be a succinate (or propionate)CoA ligase, or a novel (biotin-independent) propionyl-CoA carboxylase.
pSB1C3-ygfH(EocRI PstI）	This gene encode propionyl-CoA:succinate CoA transferase that catalyzes a CoA transferase reaction from propionyl-CoA to succinyl, generating propionate.
pSB1C3-ygfG(EocRI PstI）	The third gene in the operon encoding methylmalonyl-CoA decarboxylase that catalyzes the decarboxylation of methylmalonyl-CoA to propionyl-CoA
pSB1C3-sbm(EocRI PstI）	Sbm encodes methylmalonyl-CoA epimerase which catalyzes the reversible reaction of succinyl-CoA and methylmalonyl -CoA

2 .In the synthetic network, we found that propionate’s throughput can rise over 10% as the copy number of ygfD and sbm increase, yet over 5% rising as the copy number of ygfH and ygfG increase.
As to the oscillator device, we were happy to see the dual-feedback circuit driven by the hybrid promoter was constructed. But we can still did not observe the periodically changing fluorescence during the fermentation.To testify the hypothesis of the modeling team, we add LAA-tag to each gene in the oscillator device, we successfully extended the period to 1 hour.

Future work

There is a big challenge in our project that the recombination microbe has to be validated in the human intestine. Based on the pre-existing work, a plenty of works are coming to us in the near future.
1.Regulating the period of propionate utilizing the frequency divider with a ssrA-tag analog attached to the end of enzyme.
2. Replace the report gene rfp encoding red fluorescence protein with key gene encoding enzymes in the synthetic pathway.
3. As we know that E.coli is the most popular chassi used in the synthetic biology. But according to the people we sent questionnaire to, they prefer eating food containing probiotics rather than eating bacteria. So we are going to transform the regulatory net into bifidobacterium, which enjoy a highly reputation among the dairy industry. We will measure the propionate outside of the human body.
4. Using mathematical modeling to imitate the environment in the intestine and the concentration decreasing of propionate in the blood circulation.

Judging Critieria

Already registered in the official website in 13th March and was accepted in 12th April.
We completed safety form, judging form and team wiki before the deadline. It is for sure that we are going to present a poster and a talk at the iGEM Jamboree.
We documented four newly standard BioBrick Part(sbm/ygfG/ygfH/ygfD) used in our project and submitted them to the iGEM Registry adhere to guidelines.
Our works aims at maintaining the blood pressure through microbe metabolism SCFA, which is a new application in medicine to our knowledge.
We did plenty of experiment to validate that two of BioBrick Part of our own design and construction works as expected.
We share information with WHU and HZAU and help with each other on characterizing one part.