Abstract

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.

Oscillator

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.

Propionate

reactor

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.

Future work

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.

Protocol

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.

Modeling

Detailed analysis of the oscillator makes us clearer about how it works. From the establishment of DDEs (Delayed Differential Equations) to parameter sweep and sensitivity analysis, we know how each parameter contributes to the period. The MCOS (Multi Cells Oscillation Simulation) shows us the feasibility of a group of oscillators that can be eventually applied in practice in vivo (colon).

Human

practice

Human practice:We have done a remarkable job in intro- ducing 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 a public speech in our school about what we achieved,sharing the feelings in the iGEM competition.

Team:HUST-China

From 2013.igem.org

Abstract

Acknowledgement