Team:USTC CHINA/Project/Design

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Gene Circuit

Introduction

Our T-vaccine consists of four modules, each of which carried a gene circuit independently. With an excellent transdermal peptide TD1, three of the engineering B.subtilis could express a series of fusion proteins. The first one expresses TD1-Antigen fusion proteins, as the core of our vaccine. The second one expresses TD1-Anjuvant fusion proteins, which enhance the antigenicity. The third one is the reporter, which notify users when the status of vaccine patch is all right to be sticked to arms. The last module is a kill switch,which is designed as a reliable suicide system in bacillus subtilis to prevent it from spreading for the very first time in iGEM. We distributed different gene circuits to different bacteria(Modularization idea), by changing the ratio of each bacteria we could make it compatible with any vaccine.

Work Flow

Our Design

1. Antigen

As the main part of T-vaccine, TD-1 is fused with different kinds of antigens, so it can penetrate the skin and provoke immune responses. We have designed three kinds of vaccine, which are against hepatitis B, tuberculosis and anthrax. Our ELISA test had proved their antigenicity and mice test will figure it out if they bave immunogenicity,thereby proving that T-vaccine can be generally used.



2. Adjuvant:LTB

Similar with traditional injection vaccine, adjuvant should be added into vaccine to ensure immunity. We fused TD-1 with LTB. LT has adjuvant activity and can assist foreign antigen to induce the body to produce systemic immune response. The B submit of LTB protein has no toxic effect and it also works.



3. Adjuvant:TNFα

TNFα can recruit Langerhans cell(LC) which work as antigen-presenting cells around epidermis, and improved LC could transmit into adjacent lymph node provoking the immune response. Because this circuit is quite similar with 1# and 2# ,while its effect might be difficult to certified, so we delayed the schedule of this part. 3# is the only circuit which has not been built or tested during the whole summer experiment.

4. Reporter System

To be more user-friendly, 4# contains a reporting system. After melting in water, the spores will germinate and express blue pigment protein (amilCP) to report the best using time. Meanwhile, 4# could also ensure biosafety, as 4# engineering bacteria can kill all the engineered bacteria after use.

4.1 Reporter

T-vaccine is also a user-friendly product. Our potential users are not medical professionals, they activate T-vaccine by exposing it to water, and the patch gives visualized signal to notify users whether the status of engineered bacteria is all right and when to stick the patch to arms. We achieve this simply with the regulation of promoter 43, which is recognized by sigma factor A. The activity of promoter 43 is maximal during the exponential growth phase. In other words, we expect engineering Bacillus subtilis to express blue pigment a few hours before expressing massive antigens and adjuvants. It is the perfect time.



4.2 Kill Switch



Cells of B. subtilis enter the pathway to sporulate under conditions of nutrient limitation but delay becoming committed to spore formation by killing nonsporulating siblings and feeding on the dead cells. Killing is mediated by the exported toxic protein SdpC. Extracellular SdpC induces the synthesis of an immunity protein, SdpI, that protects toxin-producing cells from being killed. SdpI, a polytopic membrane protein, is encoded by a two-gene operon under sporulation control that contains the gene for an autorepressor, SdpR. The autorepressor binds to and blocks the promoter for the operon. Evidence indicates that SdpI is also a signal-transduction protein that responds to the SdpC toxin by sequestering the SdpR autorepressor at the membrane. Sequestration relieves repression and stimulates synthesis of immunity protein. The kill switch is based on a high-copy vector fused with promoter for operon sdpIR and coding sequence for protein SdpC. When SdpC toxins are sensed,they will be captured by Immunity Protein SdpI at the membrane, enabling SdpI to sequester SdpR. As a result, repression on promoter SdpIR is released and more SdpC will be produced. Trapped in this endless loop, the SdpC producing cells fail to cope with enormous toxin SdpC and doomed after eliminating their siblings. Eventually, the group of engineered B.subtilis is destroyed instead of sporulating.

We Also designed a test circuit, which contains promotor grac and sdpABC only, aiming to determine the ability of SdpC.



References

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