Safety With might and main
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. Bidirectional 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.1)
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[2]. 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.
Q&A
1. If your product is successful, who will receive benefits and who will be harmed?
If our project can be applied to real life, it will benefit the farmers a lot firstly because our engineering bacteria are able to trap and kill the nematodes efficiently and conveniently. Next it is beneficial to the creatures who live in the environment suffering serious nematodes disasters because the traditional nematicides do harm to human. In addition our device is easy to operate and manage. It is suitable for the burst of the nematodes disasters. So the government and the related department can benefit a lot.
2. What happens when it's all used up? Will it be sterilized, discarded, or recycled?
Our device is designed to be recyclable so the growing state can be checked at regular intervals, which means once something wrong happens, we can adjust the bacterial colony timely. In addition, after the engineering bacteria works, the device would be recycled and the residual bacteria would be disinfected.
3. Is it safer, cheaper, or better than other technologies that do the same thing?
We concluded the action objects and disadvantages of various traditional nematicides of China according to related literatures, and the result is shown below.
| Nematicide | Kinds of the nematodes it kills | Disadvantages | ||
|---|---|---|---|---|
| Fumigant | halohydrocarbon | Halohydrocarbon nemafene | Kill the nematodes in the soil and other small pests | High toxicity Do harm to the soil, the environment even the health. The using period is limited. It is probably do harm to the crops. Preventive nematicides. The operation is very complex. |
| Ethylene dibromide | ||||
| Dibromochloropropane | ||||
| Isomethylthiocyanate releasing agent | Isomethylthiocyanate releasing agent | Use as biocide, kill the nematodes, the grass and so on | ||
| non–fumigant | organophosphorus | Dichlofenthion | Apelenchoides fragariae and most of the nematodes | Have toxicity The underground water will be polluted. |
| Ethoprophos | ||||
| Isofenphos-methyl | ||||
| Isazofos | ||||
| oxamyl | ||||
| fenamiphos | ||||
| the pesticide of the biological source | Abamectin | Meloidogyne; Pratylenchus; Radopholus; Tylenchulus; Heterodera glycines | Very expensive | |
| biological agent DiT | Meloidogyne; Heterodera; Pratyleachus spp.; Trichodorus spp.; Belonolaimus spp. | |||
- Zhang C, Ye B C. Real-time measurement of quorum-sensing signal autoinducer 3OC6HSL by a FRET-based nanosensor[J]. Bioprocess and biosystems engineering, 2014, 37(5): 849-855.
- Zhang Y, Zhang J, Hoeflich K P, et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli[J]. Molecular cell, 2003, 12(4): 913-923.