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 mimicking the electronic combination 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.

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.

Synthetic biology is a real interdisciplinary field, involving chemists, biologists, engineers, physicists or computer scientists [1]. As an emerging discipline, synthetic biology really brought us a lots of challenges and opportunities. However, plenty of problems also occurred in society.

The biggest problems are biosecurity and biosafety.

According to the WHO (2004), biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release. Since the 9/11 event the funding for work on biodefense has dramatically increased in the US. The US Government Civilian Biodefense Funding, between fiscal year 2001 and 2008 cost US tax payers more than 39 billion $. And here comes a line chart of a website which is related to the bio-safety. We can easily find that populaces are focusing on this area more and more, which should, and already caused our attention.

What’s more, economy is also a big problem of synthetic biology. According to the same organization, biosecurity is the prevention of loss, theft, misuse, diversion orientational release of pathogens and toxins. Nowadays, entrepreneurs are facing the threat of theft, and thus, we also focused on this problem.

Biosafety

Have you ever played Plague Inc.? If the answer is “yes”, you sure have experienced the horror of a organisms destroying our whole world. As we all know, industrial bacterial can secrete several chemicals that has considerable significance for us. However, they are also easily stolen by thieves. What’s more, the pathogenic of bacteria can’t be depressed at this time. The threat of bio-weapon cannot be ignored

In 2003, CIA has made a report in development of bioweapons. They assessed that “Industrial biological products might be horrible than any disease that we already known”. In 2006, DSAC (Defense Science Advisory Council) also started a survey of military opportunities and challenges in this area [2].

For example, many bioterrorism incidents have occurred in the whole world wide. Such as the anthrax mail attack which is occurred after 9/11.It was said that these anthrax strains were from a laboratory. Which means it’s necessary for us to develop some method to contain microorganisms inside the lab.

What’s more, to limit the emission of synthetic microorganisms to the environment, we also need a method to prevent it. There is no reason to believe that full biosynthesis of currently semi-synthetic drugs such as heroin or cocaine, or fully synthetic amphetamine-type stimulants will not be possible and economically attractive using the toolkit of synthetic biology in the near future.

Recent years, it is reported that a new form of biology is rising: “biohackery” [3]. Biohackery allows everyone operation the genome of bacteria in their home or garage with themselves. Without the regulatory of an International Association of Synthetic Biology, we can’t make sure that there is no threat coming from someone’s house or garden, isn’t it?

The interactions between environment and the microorganisms are also an important application of our project. If the “grey goo” disaster breaks out, we can provide a modus to minimize the harm.

Biosecurity

In economy, there are a number of probiotic microbes being used in the manufacturing of various food, biofuels and pharmaceutical products. In biotech 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.

In contrast to a biohackery scenario that is driven largely by curiosity, another scenario enabled by the availability of this technology may involve illicit economic purposes.

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.

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.

3. REFERENCE

[1] Schmidt M. Diffusion of synthetic biology: a challenge to biosafety[J]. Systems and synthetic biology, 2008, 2(1-2): 1-6.

[2] Balmer A, Martin P. Synthetic biology. Social and ethical challenges. An independent review commissioned by the Biotechnology and Biological Sciences Research Council (BBSRC)[J]. Swindon, UK: BBSRC, 2008.

[3] Bennett G, Gilman N, Stavrianakis A, et al. From synthetic biology to biohacking: are we prepared?[J]. Nature Biotechnology, 2009, 27(12): 1109-1111.

[4] Ball P. Synthetic biology for nanotechnology [J]. Nanotechnology, 2005, 16(1): R1.

1.1 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 Fig.1-1.

We designed a transcriptional 2-input AND gate, in which one input promoter drives the expression of an activator and the second input promoter drives the expression of a chaperone protein. The chaperone is required by the activator to turn on the output promoter. The protein–protein and protein–DNA interactions is shown in Fig.1-2.

1.2 IDEA AND DESIGN

We all understand that logic gates must meet our requirements. Thus we used some clever ways to construct them. 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 Fig.1-3.The system is a multilayer logic gates composed by a variety of signaling molecules, which can simulate digital locks or electronic locks. This system consists of three Sensors, an integrated circuit and a reporter gene.

Firstly, We artificially synthesised the genes of ipgC(BBa_K1325000),mxiE(BBa_K1325001),sicA*(BBa_K1325002),invF(BBa_K1325003),pipaH*(BBa_K1325004) and psicA (BBa_K1325005). The agarose gel electrophoresis analysis of gene ipgC, mxiE, sicA*, invF, pipaH* and psicA is shown in Fig.1-4.

Secondly, we assembled three Sensors and the agarose gel electrophoresis analysis of Sensor A,Sensor B,Sensor C is shown in Fig.1-5.

We verified Sensor A, Sensor B and Sensor C through Fluorescence microscope. The fluorescence photos are shown in Fig.1-6.

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.

1.3 RESULTS

We use two circuits to carry three Sensors, circuit one is made up of Sensor A and Sensor B, circuit two is made up of Sensor C. We have constructed two circuits and verified the function of the first circuit. Two circuits are shown in Fig.1-7 and the agarose gel electrophoresis analysis of the two circuits is shown in Fig.1-8.We verified circuit one after 6h induction through fluorescence activated cell sorter (FACS) and fluorescence microscope. The results are shown in Fig.1-9 and Fig.1-10.

We verified circuit one after 6h induction through fluorescence activated cell sorter (FACS) and fluorescence microscope. The results are shown in Fig.1-9 and Fig.1-10.

Unfortunately, the FACS result show that although lower than the sample which add Ara and IPTG, the control also have very high fluorescence intensity，we asked Tae Seok Moon, an assistant professor in Washington University, he told us that pipaH* has a very high basal level, so it should be connected to engineered RBS to fine-tune expression from it. So we changed the RBS from B0034 to J61100 because the latter have the lower bound strength，the results are verifying.

1.4 REFERENCE

[1] Moon T S, Lou C, Tamsir A, et al. Genetic programs constructed from layered logic gates in single cells [J]. Nature, 2012, 491(7423): 249-253.

2.1 INTRODUCTION

In order to enter passwords sequentially, we designed a password-control system by engineering artificial small RNAs for conditional gene silencing in E.co-Lock. A majority of the trans-acting sRNAs in bacteria interact with the untranslated region (UTR) and the translation initiation region of the targeted mRNAs via imperfect base pairing, resulting in reduced translation efficiency and mRNA stability. Additionally, bacterial sRNAs often contain distinct scaffolds that recruit RNA chaperones such as Hfq to facilitate gene regulation (Fig.2-1).

In this study, we describe a strategy to engineer artificial sRNAs that can regulate desired genes in E.coli. Using LacZ as reporter gene that was fused to a native 30 mRNA leader sequence, active artificial sRNAs were screened from libraries in which natural sRNA scaffolds were fused to a designed antisense domain. Artificial sRNAs that posttranscriptionally repress two genes ipgC and sicA were isolated and characterized.

We anticipate that the artificial sRNAs will be useful for dynamic control and fine-tuning of endogenous gene expression in bacteria for applications in synthetic biology.

2.2 IDEA AND DESIGN

1.Construction of small RNAs regulatory system. The system is composed of box1 and box2. The model figure is below.

The process of design small RNAs regulatory plasmid

1)Construction of small RNAs regulatory system. The system is composed of box1 and box2. The model figure is below.

2)．Screening of function silencing sequences.

(a)Add target-gene and anti-target-gene sequence to small RNAs regulatory system by One-step Site-directed Mutagenesis.

(b)Verify the effect of silence.

In our protocol, we design two kinds of sRNAs, including sRNA-sicA and sRNA-ipgC. Based on the reference “Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli[1]”, we try to design sRNA-sicA for 24nt and sRNA-ipgC for 15nt. Their secondary structure figures is as follow. Now, we will introduce how to explore the inhibition efficiency of the sRNA.

We constructed two boxes in the PSB1C3 plasmid, the gene in the box1 could express normally the sRNA which inhibit the target gene in the box2.So we could measure the enzyme activity of the β-galactosidase which expressed by the lacZ gene to explore the inhibition efficiency of the sRNA we design.

According to the Michaelis-Menten equation of the enzyme kinetics, we found the best training time of the second seed is 5.5 hours and the best reactivity time after adding ONPG is 30 min in the process of the β-galactosidase assay.

As you see, the inhibition efficiency of the sRNA-ipgC is about 60% and the sRNA-sicA is about 35%. Apparently, the sRNA-ipgC has better function than the sRNA-sicA.But both of them have function of inhibition. Next step, we will continue to explore their structure to get better results.

2.3 REFERENCE:

[1] Seung Min Yoo, Dokyun Na & Sang Yup Lee1. Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nature (2013)

3.1 INTRODUCTION

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 [1]. Additionally, MinE is a topological factor.

Under normal conditions, MinC and MinD act in concert to form an inhibitor of cell division (Fig.3-1(a)). When combined with MinC, MinD increases the activity of MinC by 25~50 times [2]. The MinC and MinD division inhibitor is required to prevent separation at the potential division sites at the cell poles by preventing formation of the FtsZ ring [3]. However, the MinC and MinD division inhibitor lacks site specificity without MinE, for it prevents separation at all division sites——both polar and central (Fig.3-1(b)). The role of MinE is to give topological specificity to the MinC and MinD division inhibitor [4]. 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.

3.2 IDEA AND DESIGN

If we want to protect our special strains get rid of the theft threat, 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. So we designed a device to allow the cells grow at low density when the Eco-Lock is closed and at high density when the Eco-Lock is open.

As described in the gene circuit, when add inducer(AHL) 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 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 AHL, 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.

3.3 RESULTS

Min system contains three functional genes, MinC ,MinD and MinE . MinC is cell division inhibitor of the MinC - MinD -MinE and DicB - MinC systems that regulate septum placement. MinD is membrane ATPase of the MinC - MinD - MinE system that regulates septum placement .MinE is cell division topological specificity factor .We designed 3 pairs of primers to copy MinC, MinD and MinE from E.coli which contained the enzyme digest sites of EcoRI, XbaI, spaI and PstI . MinC, MinD, MinE were constructed into pSB1C3 as new standard parts. We constructed MinC and MinD in this part. We obtained standard biological parts J23119 - B0034, C0060, J23119 - B0034 - C0062, R0063, B0032 from iGEM, and we finished the ligation of the whole circuits of Min system through 3A assembly and OE PCR. While the verification of this section is still in progress.

REFERENCES

[1]Piet A,Robin E.Roles of MinC and MinD in the site-specific septation block media ted by the MinCDE system of Escherichia coli.Journal of Bacteriology,1992,(1)

[2]S L ROWLAND, X FU.Membrane Redistribution of the Escherichia coli MinD Protein  Induced by MinE.Journal of Bacteriology,2000,(3)

[3]Xuan-Chuan Yu , William Margolin.FtsZ ring clusters in min and partition mutants: roleof both  the Min system and the nucleoid in regulating FtsZ ring localization. Molecular Microbiology, 1999, (2)

[4]Karsten Kruse, Martin Howard.An experimentalist’s guide to computational modeling of the Min system. Molecular Microbiology, 2007,(5)

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