Introduction

T-vaccine consists of four modules, each of which carries 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 notifies users when the vaccine patch is ready to stick. 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), and 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 to penetrate the skin and provoke immune responses. We have designed three kinds of vaccines against hepatitis B, tuberculosis and anthrax respectively. Our ELISA test had proved their antigenicity and mice test will figure it out if they have immunogenicity, thereby proving the practicality of T-vaccine.

2. Adjuvant:LTB

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

3. Adjuvant:TNFα

TNFα can recruit Langerhans cells(LC), which work as antigen-presenting cells around epidermis, and improve LC could transmit into adjacent lymph node to provoke immune responses. This circuit is quite similar to 1# and 2# , yet its effect might be difficult to certify, which put off 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. Melting in water, the spores will germinate and express blue pigment protein (amilCP) to report the optimal using time. Meanwhile, 4# could also ensure biosafety by killing all the engineered bacteria after usage.

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.

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, which 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

1.Kang, T.J., et al., Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization. Transgenic Res, 2003. 12(6): p. 683-91.
2.Mrsny, R.J., et al., Bacterial toxins as tools for mucosal vaccination. Drug Discovery Today, 2002. 7(4): p. 247-258.
3."Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis."Belisle J.T., Vissa V.D., Sievert T., Takayama K., Brennan P.J., Besra G.S. Science 276:1420-1422(1997)
4."Evidence for a partial redundancy of the fibronectin-binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis." Puech V., Guilhot C., Perez E., Tropis M., Armitige L.Y., Gicquel B., Daffe M.Mol. Microbiol. 44:1109-1122(2002)
5."The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity." Singh Y., Klimpel K.R., Quinn C.P., Chaudhary V.K., Leppla S.H.J. Biol. Chem. 266:15493-15497(1991)
6."Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin." Varughese M., Teixeira A.V., Liu S., Leppla S.H. Infect. Immun. 67:1860-1865(1999)
7.Blumberg B, Alter H (1965). "A "new" antigen in leukemia sera". JAMA 191: 101–106.
8.Tiollais P, Christine P, Dejean A. The hepatitis B virus.Nature, 1985,371: 489.