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 promoter. It is activated by the AraC protein in the pre- sence of arabinose and repressed by the LacI protein in the absence of IPTG. Hence, activation of the promoters by the addition of Arabinose and IPTG to the medium r- esults in transcription of each component of the circuit. Increased production of AraC in the presence of arabin- ose results in a positive feedback loop that increases p- romoter activity. However, the concurrent increase in production of LacI results in a linked negative feedback loop that decreases promoter activity. The differential activity of the two feedback loops can drive oscillatory behavior.

Propionate

reactor

A four gene operon in E.coli K12 genome which includes sbm ygfG ygfH ygfD, is significant in the metabolic pathway that converts succinate to propionate through Wood-Werkman reaction. We construct effective expression plasmid to increase the quantity of the four enzymes independently. By detecting the propionate amount by HPLC, we get to know which of the four is the rate-limiting enzyme. We choose it as the output of the oscillator.

Future work

In the future, we will use the gene of rate-limiting enzyme of propionate producing reaction to replace mRFP in the oscillator as the output. And we hope to see periodical r- elease of propionate. Later ,we may use mouse model to demonstrate our original design.

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 (MultiCell Oscillation Simulation)shows us the exact feasibility of the oscillator both in vitro and in vivo (colon).

Human

practice

Human practice:We have done a remarkable job in intro- ducing high school students to 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 DNA materials and characterizing their parts. Furthermore, we made a public speech in our school to report what we achieved, to sha- re the feelings in the iGEM competition.And we also desi- gn a crossword puzzle about iGEM for fun of all iGEMers.

Team:HUST-China

From 2013.igem.org

Abstract