Team:NAU-CHINA/Composite Part

• BBa_K3408006
• BBa_K3408007
• BBa_K3408008
• BBa_K3408009
• BBa_K34080010
• BBa_K34080011

Overview

To initially verify that our system can finally serve its function, we need verifications of different composite parts. Afterwards we can assemble them to build our engineering system. So, in this year, we designed six progressive composite parts to verify some critical functions of our system. Due to the influence of COVID-19 this year, all our verifications are based on the literature and mathematical theory.

P_nar-RBS-GFP- terminator

Part P_nar is an oxygen-free inducible promoter and is triggered by global regulator FNR, which is induced by ResD/E two-component regulatory system. The intestine is an anaerobic environment. ResE functions as the oxygen sensor which would be active in the absence of oxygen. Then ResD would be phosphorylated by ResE to activate P_fnr and express FNR. Therefore, P_nar can be activated by oxygen-free condition to express phy(ycD). We employed this part to regulate different behaviors of our engineered Bacillus subtilis. Thus, we designed this composite part (BBa_K3408006) to verify function of the part P_nar. We added a gfp gene following the P_nar. If our part P_nar can function normally, we can see GFP successfully expressed in our Bacillus subtilis.

P_nar-RBS-phy(ycD)-terminator

Our project aims to secrete phytase to immobilize lead ions. Thus, we need to ensure that our system can secrete phytase normally. Our first composite part has demonstrated the function of part P_nar. So next step, we need to verify whether part P_nar and phytase phy(ycD) can achieve a successful assembly in this composite part (BBa_K3408007).

When our engineered bacteria are inoculated into the intestine of earthworm, under the regulation of ResD/E two-component regulatory system, it can secrete phy(ycD) to tackle with lead ions. In our experiment, we will create an anaerobic environment to verify whether our engineered bacteria can function normally to immobilize lead ions or not.

P_nar-RBS-CⅠ-terminator-P_CⅠ-RBS-GFP-terminator

On the basis of successful verification of part P_nar, we can verify effectiveness of repressor protein CⅠ, which connects P_nar and toehold switch. Successful verification of this composite part (BBa_K3408008) can well consolidate our more complicated part below.

In earthworm’s intestine, CⅠ protein will be produced to inhibit P _CⅠ to avoid expression of gene downstream. So, we used GFP as the reporter protein and designed experiment to check if the CⅠ/P _CⅠ could work successfully.

P_liaG-trigger RNA-terminator-P_CⅠ-switch RNA-GFP-terminator

Our toehold switch comprising of trigger RNA and switch RNA is significant to our overall design of genetic circuit. So, it’s essential to verify the feasibility of this part. Thus, we added gfp gene following the toehold switch.

In our experiment, we used P_liaG and P_CⅠ to control the transcription of trigger RNA and switch RNA. By virtue of this composite part (BBa_K3408009), we could verify if our toehold switch works normally.

P_nar-trigger RNA-terminator-P_CⅠ-switch RNA-mazF-terminator

Now we have verified our P_nar, optimized CⅠ and toehold switch. Further verification of more complicated assembly of our parts can lay the foundation of our future demonstration of the whole system. Considering the bio-safety, we will let our engineered bacteria commit suicide by expressing mazF. Based on this purpose, we try to achieve a good assembly of P_nar, toehold switch and toxin protein MazF.

When in the intestine, Bacillus subtilis will accumulate trigger RNA. With time going by, engineered bacteria will be discharged outside and switch RNA will be transcribed with the existence of oxygen, so MazF could be translated and kill engineered bacteria. In our experiment, we will respectively create an anaerobic and an aerobic environment to check if our parts could assemble excellently. Follow-up experiments will verify the effect of MazF.

Successful verification of this composite part (BBa_K3408010) can be very helpful to the overall building of our whole engineering system.

P_liaG-RBS-lacI-P_grac-RBS-CⅠ-P_CⅠ-RBS -GFP-terminator

In our design, to guarantee successful culture of our engineered Bacillus subtilis, we introduced an IPTG-inducible system in our bacteria. Thus, this composite part (BBa_K3408011) is aimed to demonstrate that the IPTG-inducible system can actually work in our Bacillus subtilis.

LacI is the transcriptional repressor protein, which could bind to the specific site of P_grac to inhibit activity of P_grac. IPTG can break out this inhibition by prior combination with LacI. So, there’s no expression of GFP in Bacillus subtilis.