Hypertension has become the leading risk factor for mortality worldwide. Human’s blood pressure (BP) has a basic daily rhythm with two peaks,6:00 to 10:00 in the morning and 16:00 to18:00 in the afternoon. The morning peak is also called “death time”,for it’s unlikely to take drugs before waking. Propionate, a short chain fatty acid, was recently shown to produce an acute hypotensive response.2013 HUST-China iGEM use a synthetic way to combine bio-oscillator with propionate-producing enzyme gene, trying to build a gut probiotic which can release propionate periodically in accord with the rhythm of human BP. This could be a great substitute for chemical drugs by saving patients from drug dependence and the risk of sudden death at morning BP peak time.
The key part of the oscillator is araBAD/ lacZYA hybrid p- romoter. It is activated by the AraC protein in the prese- nce of arabinose and repressed by the LacI protein in the absence of IPTG, constructing two feedback loops with opposite effects.And the differential activity of the two feedback loops can drive oscillatory behaviour.
A four-gene operon in E.coli K12 genome which includes sbm,ygfG,ygfH and ygfD, is significant in the metabolic pathway that converts succinate to propionate through Wood-Werkman reaction. We constructed effective expression plasmid to increase the quantity of the four enzymes independently. By measuring the propionate amount, we figured out which of the four is the most effective.
In the near future, we will regulate the period of propionate utilizing the frequency divider with a ssrA-tag analog attached to the end of enzyme.Besides,we will replace mRFP with key gene in the synthetic pathway, hoping to see periodical release of propionate in accord with the rhythm of human BP.
Our project is divided into three parts: the construction of the biological oscillator, the output evaluation of propionate by HPLC and the standardization of four genes.
Detailed analysis of the oscillator makes us clearer about how it works. From the establishment of DDEs to parameter sweep and sensitivity analysis, we know how each parameter contributes to the period. The MCOS shows us the feasibility of a group of oscillators that can be eventually applied in practice in vivo (colon).Lastly, we statistically analyzed our simulated data and wet-lab data.
We have done a remarkable job in introducing high school students synthetic biology and iGEM jamborees as well as motivating them towards future participation in the iGEM. Besides, we collaborated with two other iGEM teams by sharing plasmids and characterizing their parts. Furthermore, we made public speeches in our school about what we achieved,shared the felt in the iGEM competition.