Team:HZAU-China/Project/The design of experiment

• Overview
• The innovation of HZAU-iGEM Team
• Rabies
• Flea and Yersinia pestis
• Bacterial Spores as Vaccine Vehicles
• Bacillus subtilis as Probiotics
• Antimicrobial Peptides
• The design of experiment

We chose Bacillus subtilis as our chassis, because it has some characters that are very suitable for a “safe moving vaccine factory”.

The following are the reasons:

1.Bacillus subtilis can form biofilm that we think is important for being transferred from fleas to dogs.

2.Bacillus subtilis has many advantages as a tool for vaccine vector.

3.Bacillus subtilis has already been used as probiotics and maybe exists in the internal environment of fleas.

The experiment designs and genetic circuit are as follows:

1.For the vaccine question

In order to know whether our vaccine can activate the immunity system of dogs, we used B.subtilis as vaccine vector to express secreted glycoprotein. Five proteins are present in rabies virus particles: nucleoprotein, phosphoprotein, matrixprotein, glycoprotein and RNA-dependent RNA polymerase. Because of containing the neutralizing epitopes which are the targets of vaccine-induced immunity, the glycoprotein can stimulate the organism to produce antibody against Rabies.

2.For the moving question

In order to know whether the modified B.subtilis can be regurgitated by flea, two experiments are designed One is to feed flea using modified B.subtilis that can express GFP. This design is to know whether the flea can eat B.subtilis that colonizes in the proventricular. Another goal is to test whether the bacteria can make flea vomit.

'''''Notice''''': We stopped this experiment because of safety reason. We haven’t found safe flea to achieve this experiment. Although we currently cannot provide experimental data to solve this question, we are ready to provide a complete experiment protocol and the devices needed in the experiment. In the future, when we make sure it is safe enough to release flea, we will cooperate with professional institutes to finish it.

3.For the safety question

A certain number of infectious bacteria carried by flea will cause potential risks which are hard to control, so broad spectrum Antimicrobial Peptides have been designed as secreted protein to kill those infectious bacteria such as Yersinia pestis.