Gene Circuit

Introduction

Our T-vaccine consists four modules. Each of them carries a gene circuit independently. The first one expresses TD1-Antigen fusion protein, as the core of our vaccine. The second one expresses TD1-Anjuvant fusion proteins, which enhance the antigenicity. The third one is reporter, it notifies users whether the status of vaccine patch is all right and when they can stick the patches to arms. The last module is a kill switch，we designed a reliable suicide system in bacillus subtilis for the very first time in iGEM. So it would be OK if our users tear patches off and throw them away. We attribute different gene circuits to different bacteria(Modularization idea), by changing the ratio of each bacteria we could make it compatible with any vaccine.

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

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. 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

In B.subtilis, when it comes to the stationary phase, the environmental pressure increases and nutrition becomes limited, so B.subtilis begins to produce spore. Now the community will be divided into two different parts. One of them are trying to kill others to get enough nutrient, delaying the production of spores and achieving a competitive advantage. Killing is mediated by the exported toxic protein SdpC. SdpI will appear on the membrane surface to avoid itself from being damaged. SdpI could bind free SdpC and autopressor SdpR, to remove inhibition of SdpR against I and R, to produce more SdpI to offset SdpC, finally guaranteeing the subgroup alive, thereby delaying the spores production.We transfer SdpC which is fused by promoter SdpI/R into high copy plasmids in order to damage the balance of the system, thereby killing whole colony. When SdpC appears, SdpI on the membrane will bind free SdpC and adsorb SdpR to cease its inhibition against SdpI P/R, trying to produce more SdpI. At the same time, it will activate the promoter SdpR/I in our circuits and generate more SdpC. The system would fall into an infinite loop, and according to our modeling,the amount of SdpC increases beyond the ability of SdpI. Thus, the cells with protection mechanism will crack and die because of too much SdpC. All of them formed the killing device.