Team:RDFZ-China/Project Description

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• • What are we facing?
  • Predecessors
  • Project Xscape
  • For Fermentation
  • For Therapy
  • Metabolic Stress
  • DIY Bio and Biosafety
  • Community and Future
  • References

What are we facing?

Biosafety has always been the major concern to the public, to the companies and the researchers. Doubts and worries raised just as genetic technology was invented. With the rapidly growing of synthetic biology and iGEM community, more and more synthetic biology products are built with the widely distributed DNA toolkits or the inexpensive DNA synthesis service(Synthetic and Will); we are facing unprecedented biosafety issue that unwanted leakage of synthetic biology products to the environment may cause an unexpected but definitely disastrous problem.

Predecessors

For decades, researchers were striving to build biosafety devices through auxotrophy or external inducive kill switches(Lee et al.), holins and restriction enzymes are most commonly used. Most of the failures of the previous devices were caused by mutation and evolution of immune(Moe-Behrens et al.) .

The two major threats of engineered microbes’ leakage are the possible Horizontal Gene Transfer which will lead to the spread of recombinant DNA to the entire ecosystem, or the engineered bacteria could contaminate or overrun the natural habitat.(Wright et al.)

Project Xscape

Under this circumstance, this year we decided to be a fundamentalist to synthetic biology, by using genetic circuits and logic gates, to establish biosafety devices which can apply to the real-world situation.

Since cell death and lysis mean there is a continual presence of free DNA in the environment, holins, which are most widely used are excluded from our choices, and colicin E2 nucleases (Darmstadt iGEM2016) came into our site. We choose site non-specific nucleases since the entire genome and plasmids needed to be entirely digested to prevent the spread, and we use nucleases from a different family to prevent the possible evolution of nuclease inhibitors. Artificial DNA, RNA, and amino acids are a good solution, but due to its high cost so far, it is not applicable to most of the user.

For fermentation

The first device we build is for the fermentation; we want to execute the escaped engineered bacteria from the fermenter, accidentally or intentionally. We used two environment factors to monitor the bacteria’s situation: temperature and population density, they are both high and tunable in the fermenter. So, the device will initiate when temperature and density are both low. We used thermal sensitive regulator (NUS iGEM2017)(Piraner et al.) and quorum sensing regulator (MIT iGEM2004) (Canton et al.)as our sensor, sRNA(Storz et al.) and tetR family repressor PhlF(Glasgow iGEM2015)(Stanton et al.) as the signal inverter. We add intergrase (Peking iGEM2017) controlled by the thermal sensitive regulator, which will turn the promoter of a lethal gene when temperature rise in the fermenter so that bacteria can survive at the very beginning. Also, we build a model to stimulate the minimum autoinducer required at the beginning of the fermentation, same as the purpose of integrase. This model is for keeping bacteria alive at the very beginning of fermentation. Together they form a NOR gate which will lead to cell death through genome degradation when temperature and density decrease.

For Therapy

The second device we build is for therapeutic bacteria, the device can carry out noninvasive tracing through ultrasound imaging of the gas vesicle(Shapiro et al.), release the drug (from SHSBNU 2017) controlled by a thermal sensitive regulator at nidus by ultrasound tissue heating, and heat to a higher temperature to release nuclease and kill the bacteria after it finishes its mission.

For Metabolic Stress

We applied capacity monitor (Ceroni et al.) to quantify the expression burden of all our systems, and to reduce the metabolic stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking iGEM2011) and cold-regulated 5’UTR region (Ionis Paris 2017). This device only involves one transcriptional regulator, which will be less energy consuming.

DIY bio and Biosafey

Back to the growing and glowing synthetic biology community, despite the ones doing it on campus, more and more people are starting it at home, t