Abstract

Hypertension has become the leading risk factor for mortality worldwide. Human’s blood pressure has a basic daily rhythm with two peaks, 6:00 to 10:00 in the morn- ing and 16:00 to18:00 in the afternoon.The morning peak is also called “death tim- e”,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 enzy- me gene, trying to build a gut probiotic which can release pro- pionate 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 m- orning 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 future, we will use the gene of key enzyme of propionate producing reaction to replace mRFP in the oscillator as output. And we hope to see periodical r- elease of propionate.

Protocol

Aside of traditional molecular biology method, we mainly use HPLC to detect propionate, flow cytometer(FCM) and confocal to detect fluorescence.

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 fu- ture 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,sha- ring the feelings in the iGEM competition.

Team:HUST-China

From 2013.igem.org

Abstract