Project Circuit Design
Overview
This year, our project mainly concerns the crucial pathogenic organism nematodes. We intend to build a kind of novel engineering bacteria which will express organics to allure nematodes, and synthesis specific toxin to kill the pests after they devour them. Hereby we are able to build an environmentally friendly system of killing the nematodes.
Although Bace16 and rMpL are the specific toxins of killing the nematodes, if expressed too much, they will undeniably do harm to the ecosystem of the soil itself. What’s more, taking the economic efficiency into consideration, if releasing the toxin continuously, we will not receive the maximum economic benefits. Therefore, we construct a circuit controlled by light to regulate the releasing of the toxins. The loop consists of three parts: 1. Photoinduction System; 2. Reverse System；3. Bait-Killer. (Figure 1.)
In this circuit, we achieve the following effects. When bacteria are under the light, the promoter OmpC is inhibited. Meanwhile the promoter Pcon expresses limonene constantly to allure nematodes. However, when the amount of the nematodes around is large enough, the “light ” will be turned off. At the same time, the promoter OmpR starts the expression of the int intgrase to make the promoter Pcon reverse. So the expression of the limonene turn off while the expression of the toxins start. The advantages of this loop are shown as follows:
- Steerable: we can control the expression of the inducer and the toxins by regulating the light.
- Environmentally friendly: Only when the amount of nematodes gathering around reaching a considerable point, the expression of toxins will start. Therefore, the concentration of the toxins can be limited at a reasonable range.
- Economize: The expression of toxins as well as the release of the bait are at two different stages during the trapping and killing. So we could use less E.coli to kill the nematodes.
- Avoiding of overload: By regulating the two pathways of toxin and bait, we have reduced the load of metabolism for E.coli.
Photoinduced System
cph8
The red light sensor (Cph8) is a fusion protein consisting of a phytochrome Cph1 and a histidine kinase domain, Envz-OmpR. Cph1 is the first member of the plant photoreceptor family that has been identified in bacteria. EnvZ-OmpR, a dimeric osmosensor, is a multidomain transmembrane protein. Cph1 can be inactivated under red light. Upon changes of extracellular osmolarity, EnvZ specifically phosphorylates its cognate response regulator OmpR, which, in turn, regulates the PompC. 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. While when exposed to red light, Cph1 is inactivated, inhibiting the autophosphorylation, thus turning off gene expression.
We insert RBS(BBa_B0035) and constitutive promoter(BBa_J23100) in the upstream region the Cph8 sequences(BBa_K592000) to make sure that the E.coli can express Cph8 successfully in vivo.
promoter OmpC
PompC is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC gene. Phosphorylated OmpR binds to the three operator sites and activates transcription.
We insert PompC(BBa_R0082) sequences into the upstream region of our target gene gp35 through restriction digestion and ligation, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of gp35 through the control of light.
pcyA+ho1
Moreover, in order to function well, Cph8 has to form chromophore with PCB biosynthetic genes (BBa_I15008 and BBa_I15009), where the formation of PCB requires the gene pcyA and ho1. Ho1 is a sort of heme oxygenase from Synechocystis which can oxidize the heme group using a ferredoxin cofactor, generating biliverdin IXalpha. And then, PcyA, a kind of ferredoxin oxidoreductase from Synechocystis, functions to turn biliverdin IXalpha (BV) into PCB.
We used overlap PCR to connect gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) to ensure a continuous work of the photoinduced system in our E.coli.
Bidirectional Transcriptional System
Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, and results in the reverse of the sequence（there it means the promoter Pcon） between attB and attP, changing the two sites to attL and attR at the meantime. As a consequence, the promotor moves on to express the toxin protein instead of the attractant.
We insert the gp35 gene into the downstream region of the PompC promotor by means of 3A assembly so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompC and gp35 to decrease basal integrase expression above flipping threshold level which could lower the translation efficiency of gp35. Meanwhile, we insert the recombined locus of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we have accomplished our reverse promotor system.
Bait-Killer System
We add a bait gene in the downstream region of attP at the constitutive promoter, and a gene of toxin in the dreamstream region of attB. Expression of gp35 is inhibited under light, thus the promoter turn to the original direction and express the attractant. Whereas when the light disappears, the expression of gp35 begins with the help of PompC, the direction changes to express the toxin. And in this way we established the photoinduced system.