Team:BIT-China/Project

The way we live today has been deeply revolutionized through rapid developments of low-cost drugs, novel chemicals and clean energies by various newly engineered microorganisms. However, the problem of high risks of potential ecological contamination and accidental commercial loss continues threatening the scientists, industrialists and the public as synthetic biology makes progress. Can the important strains be genetically locked up in the molecular level even when their physical lock boxes are broken up? The 2014 project of BIT-China, E.co-Lock, focused on the bio-security and bio-economy of all engineered microorganisms, is novelly designed to meet this challenge.

Layered AND Gates, sRNA regulatory System and Min System are the three main parts to build up the E.co-Lock. The layered logic AND gates are constructed and connected to realize different combinations of "the password" - here three different inducers. The sRNA regulation system is then implemented to control the order of the "password" by inhibiting the mRNAs built in the layered AND gates, thus to precisely mimic the password of a real digital lock. Currently, a three-inducer-password is built up and ideally in the future, many more layers of AND gates containing new inducers with newly synthesized sRNAs could be added up to increase the complexity of the password. Finally, Min system, representing a cell division inhibiting system, is re-designed to maintain industrial strains to an extremely low density given an initial locked state. In the future, other similar systems can be introduced to replace Min system to fulfill this function of inhibiting cell division. Therefore, commercially valuable or potentially hazardous strains will be under much more strict control with different customized E.co-Locks.

In real world, there are several examples which could prove our project is necessary. Recently, a committee of Ecological Risk Research in synthetic biology has set up in America. And there are also many laws are setting in order to restrict the accidental release of pathogens operating in the lab.

Thus, when genetically locked up with E.co-Lock, important industrial strains or dangerous strains will be under better protection from suffering loss, even at the occasions of unexpected leaking out or stolen, only when right password is put in, when suitable inducers are added in set order, strains can multiply properly. Much better safety of microorganism can be achieved both ecologically and commercially.

In economy, there are a number of probiotic microbes being used in the manufacturing of various food and pharmaceutical products. Biofuels like bioethanol and biodiesel are being manufactured using new raw materials like lingo cellulosic, algae and other biomass material to replace fossil fuels. In microbiology industry, high-quality strains are always much expensive, resulting in high frequency of theft events. This problem seriously affected the development of the whole industry.

Various synthetic gene circuits are able to provide multiple abilities to industrial bacterial. But to protect the originality result of a research group, not only the patent protection is needed, it’s also necessary to transmit a “bio-lock” into the bacteria.

Synthetic biology is a combination of nanotechnology, biotechnology and information technology . It’s easy to apply a patent but to protect it from theft is very hard.

A case on strains theft occurred in China in 2000. In this case, the strains theft caused heavy economic losses to a company. This upgraded the concerning of bio-economy problems to a new focusing level. We must take action immediately in order to protect our profit.

1.Introduction

Logic Gate, as an essential part of Synthetic biological circuit, is an application of synthetic biology where biological parts are designed to perform logical functions mimicking those in electronic networks. For example, we can refer to the state “Active” as binary number “1”, while the state “Inhibit” as “0”.We can switch those different states by adding a measurable element so that a gene circuit can be accomplished. By constructing layered logic gates, we can design a genetic lock simulating electronic ones to protect strains.

The truth table for an AND gate is shown in figure 1.

The transcription factors and chaperones were gleaned from gene clusters encoding type III secretion systems, which could be found in many pathogens. One of the best characterized is encoded within Salmonella Pathogenicity Island 1 (SPI-1)10.Within this island, it has been identified that a genetic circuit regulates the expression of exported proteins through a protein–protein feedback mechanism. The mechanism bases on two effectors, a chaperone (SicA) and a transcription factor (InvF). The SicA–InvF complex activates transcription from the sicA promoter. Thus, SPI-1 provides three parts that can form a core of an AND gate, the activator, the chaperone and the inducible promoter. The gene cluster from SPI-1 and the needle structure is shown in figure2.

2.Experiment design

We all understand that logic gates must meet our requirements. Thus we used some cleverly way to construct it. First, the gate must contain various parts in order to build multiple orthogonal gates. Second, the inputs and outputs of the gate should have a common signal carrier that enables them to be layered (the former output are expected to be the latter input). In transcriptional circuits, the inputs and the outputs are promoters. We have designed a transcriptional double-input AND gate, in which one input promoter drives the expression of an activator while the other input promoter drives the expression of a chaperone protein. The chaperone is demanded when the activator turns on the output promoter. Output promoter is active only when both input promoters are expressed. In other words, if one want to keep transcription factor active, both gate should be activated, too.

Pathway of the layered logic gate system is shown in figure 3.The system is a multilayer logic gates composed by a variety of signaling molecules, it can simulate digital locks or electronic locks. Gene in this system consists of three sensors, an integrated circuit and a reporter.

The assembly of three sensors is shown in figure 4.

We also implant the sRNA regulation system in each AND gate to repress the former one, ensuring that the signal molecule must be added in a set order, keeping the system from activated by random or total testing. Another system, cell division inhibiting system will go into effect. As a result, only with the input signals (various molecules) in correct order can permit E. coli live as expected.

3.The experimental progress

We expect to use two plasmids carrying the three Sensors, one is made up of Sensor A and Sensor B，the other made up of Sensor C.

We have already completed the construction of the first plasmid. Next work is to insert red fluorescent protein at the end of the plasmid, and at last, complete the verification of plasmid functions.

Most part of the second plasmid has been completed, but not yet connected to the Sensor C subject.

4.Expected Results

Layered logic gates form the main part of the locks by constructing two plasmids, then we can verify the function of the gene line by the addition of inducing agents. We have designed a transcriptional 2-input AND gate, in which one input promoter drives the expression of an activator while the other input promoter drives the expression of a chaperone protein. The chaperone is demanded when the activator turns on the output promoter. Output promoter is active only when both input promoters are expressed. In other words, if one want to keep transcription factor active, both gate should be activated, too.

Reference

[1] Tae Seok Moon, Chunbo Lou, Alvin Tamsir, Brynne C. Stanton1 & Christopher A. Voigt. Genetic programs constructed from layered logic gates in single cells. Nature. 2012,491:(249-253)

In order to enter the password sequentially, we introduced a plasmid password-control system by using synthetic small RNAs into our system.

The principle is: In normal condition, mRNAs combine with ribosomes and contribute to protein translation. But the synthetic small RNAs can inhibit the function of mRNA. Structure of a synthetic small RNA has a target-binding sequence and a scaffold structure sequence.

The target-binding sequence will guide the small RNA to bind to its target mRNA in the proper region--the translation initiation region. And the scaffold structure is responsible for recruiting the Hfq protein, which can help the formation of the mRNA-sRNA complex.

Thus, by replacing target sequences and target-binding sequences we can construct new synthetic regulatory sRNAs to repress downstream pathways.

To construction of small RNAs regulation system，we separated the whole method to three steps

1)Construction of small RNAs regulation box 1.

2)Construction of small RNAs regulation box 2.

3)Assemble of box 1 and box 2.

The method we chose to valid silencing sequence effect is adding target-gene and anti-target-gene sequence to small RNAs regulation system by One-day mutant method.

The sRNA system has already complete the whole construct ,what we need to do now is only to insert it into our layered logic gates.

1.Introduction

If we want to protect our special strains get rid of the stealing horror, we can’t let bacteria grow without limitation. So we must have a system to control the amount of bacteria, preventing it from industrialization after stolen.

Min system consists of MinC, MinD and MinE. MinC protein is the real division inhibitor. And MinD, as a kind of ATPase, is believed to function by activating a MinC-dependent division inhibition mechanism. Additionally, MinE is a topological factor.

Cell division in prokaryotes is initiated by the localization of the tubulin. For example, the GTPase FtsZ in the future division site. FtsZ assembles into a ring, and other proteins are then recruited to form the septal ring organelle which mediates cell envelope invagination. Division of Escherichia coli normally begins in the middle of the cell; meanwhile, the potential division sites (PDSs) present near each of the cell poles.

Under normal conditions, MinC and MinD act in concert to form an inhibitor of cell division. When combined with MinC, MinD increases the activity of MinC by 25~50 times. The MinCD division inhibitor is required to prevent separation at the potential division sites at the cell poles by preventing formation of the FtsZ ring. However, the MinCD division inhibitor lacks site specificity without MinE, for it prevents separation at all division sites——both polar and central. The role of MinE is to give topological specificity to the MinCD division inhibitor. It suppresses the action of the division inhibitor at mid of the cell, allowing FtsZ ring assembly at this site, but not at the cell poles.

2.Design（Aii）

When add inducer as input, the compound that bound of inducer and LuxR can inhibit promoter R0063. Therefore, when the cell concentration is under a certain threshold, the isolated gene minC (suicide gene) won't express in cell under normal growth. With the number of cells increasing, Aii expression increasing as well, Aii will cause the decomposition of input, leading to a lower input concentration. When the input concentration decrease to a certain threshold, promoter R0063 works, gene minC (or suicide gene) expresses, cell division stops, the number of cells stay stable.

1.A reliable way to prevent theft

2.A credible way to against accidental disclosure

3.A foolproof way to resist bio-terrorism

4.A fresh way to suppress industrial strains