Overview

To guarantee biosafety, our team took three measures as follows:

Firstly, we designed a device to cultivate E.coli inside and restricted their movement at the same time, thus preventing the difussion of our engineering bacteria to soil.

In addition, we established a photoinduced bidirectional transcription system regulated by light, where we can divide the process of attracting and killing nematodes into two periods to realize the relatively timing and quantitative release of the toxic proteins, bringing down the retention in the environment.

Thirdly, a suicide system was constructed based on the principles of quorum sensing in microorganisms which would enable us to control the bacterial colony density artificially and therefore the quantity of engineering bacteria can be maintained in a stable and controllable range.

Below indicated the specific principles and designs.

Device

Considering the biosafety, we improved device 2.0 into a relatively closed unit, and we also designed a dome to cover the medium. In addition, replacement as well as remove of medium and engineering bacteria would all be done in the labs or safety rooms of factories which guarantee relative closure of the device used in farmland and avoid engineering bacteria spreading.

Switch

Although Bace16 and rMpL are both toxic proteins towards nematodes specially, the over expression of these proteins can also break the ecological equilibrium inevitably. Therefore we build a photoinduced bidirectional transcriptional system to make the expression of toxic proteins under control so that our engineering bacteria can express attractants or toxic proteins in different conditions. This system can be divided into three main parts: photoinduced system, bidirectional transcriptional system and bait-killer system.

1. Photoinduced system

The red light sensor (Cph8) is a fusion protein which consists of a phytochrome Cph1 and a histidine kinase domain, Envz-OmpR. Cph1 is a member of the plant photoreceptor family. With the biosynthesis of PCB, Cph8 can serve as a photoreceptor that regulates gene expression through PompC. Without red light, Cph1 is activated and it enables EnvZ-OmpR to autophosphorylate which in turn activates PompC. Conversely, Cph1 is deactivated when exposed to red light, inhibiting the autophosphorylation, thus turning off gene expression. In order to regulate the direction of promoter J23110 under the light signal, an integrase, gp35, is added to the upstream region of the PompC promoter.

2. Directional transcriptional system

We constructed two main circuits. The first one expresses gp35 serine integrase,(Fig.1) which can exclusively catalyze site-specific recombination between attB and attP, the attachment sites on phage chromosome and host chromosome. This recombination results in the reverse of the sequence between attB and attP, changing the two sites to attL and attR at the meantime. This inversion can be reversible by appropriately controlling the conditional expression of integrase and an excisionase in Bxb1 named gp47 at a certain ratio.

The second one contains a switch promoter(J23110), two functional genes(there GFP and RFP), two RBS and terminators. At first, the plasmid expresses GFP. When gp35 expresses, the switch will turn around and RFP on the other side of the plasmid is going to be expressed.(Fig.2)

3. Bait-Killer system

We replaced GFP and RFP with limonene synthase and toxic protein respectively, so the release of the two proteins can be regulated by gp35 directly, and by light signals indirectly.

For more information, please see our circuit design section.

Generally speaking, the Switch system works in this way--provide the system with light of wavelength in 600nm at first so gene gp35 will be repressed and the system will express the chemical attractants like limonene. While when the concentration of attractants reaches a certain point (see modeling part), we would turn off the light to trigger the expressing of protein gp35 to push the system work in a reverse direction so that the toxic protein would be expressed. In conclusion, the significance of our system is that we have built a photoinduced bidirectional system to avoid a simultaneous and strong expression of the bait and toxic proteins, in which way we can avoid the potential harm done to environment by the system.

Suicide

We not only considered the potential safety problem caused by the high expression of the toxic protein and attractant, but also designed a suicide system for our engineering bacterias to solve the problem by regulating the population size of our bacterias.

We built our system based on the phenomenon of quorum sensing^[1]. Synthase LuxI coded by gene luxI can catalyze the synthesis of AHL, which is a kind of organic small molecule able to across the membrane freely. Also, protein LuxR encoded by gene luxR can bind with AHL molecules to form a complex. This complex can bind with promoter luxpR, after which the transcription of mazf gene in the downstream will be triggered and bacterias will be killed by toxin protein MazF. The related parts have been shown in Module 3 of our Project page previously.

Lab&Environment safety

To guarantee the lab safety, we follow safety rules strictly. For instance, all team members in the lab should wear clothes for lab-use, wear gloves and work in the super clean bench when necessary; poison reagent should be used in the fuming cupboard; and the waste liquid and medium should be poured or thrown differently.

As for environmental safety, on one hand, we design a semi-closed device to avoid the spread of engineering bacterias in which way we could increase the safety level and improve the attracting and killing efficiency of our bacteria; On the other hand, light-regulated bidirectional transcription system can avoid over-expression of toxin proteins, and the design of suicide part can regulate the population density of engineering bacteria. All in all, biosafety is ensured in both the developing and the application stage of our project.