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

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

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 bacillus 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.

Fig. 1. shows the genetic circuit of the device secreting Rabies virus strain ERA glycoprotein in bacillus subtilis. Part K541501 contains constitutive promoter veg(K143012), Ribosome Binding Site spoVG(K143021) and signal peptide SacB. iGEM11_FAGEM designed this part and it worked very good in bacillus subtilis. Part K1228002 is designed by our team. It is a rabies virus strain ERA glycoprotein coding sequence. The glycoprotein is the main protective antigen of rabies virus which can induce the body to produce neutralizing antibody.

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 bacillus subtilis that colonizes in the proventricular. Another goal is to test whether the bacteria can make flea vomit.

Fig. 2.shows the genetic circuit of GFP device. Promoter Veg is a constitutive promoter that constitutively expresses the P43 protein in bacillus subtilis .It was designed by iGEM08_Imperial_college. The rest is a GFP generator designed by Igem08_UNIPV-Pavia.

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.

Fig. 3.a shows the genetic circuit of the device secreting Human Lysozyme in bacillus subtilis. Part K541501 contains constitutive promoter veg(K143012), Ribosome Binding Site spoVG(K143021) and signal peptide SacB. iGEM11_FAGEM designed this part and it worked very good in B.subtilis. Part K122800 was designed by our team. Human lysozyme (hLZ) exhibits microbicidal activity to various degrees against several bacterial strains.

Fig. 3.b shows the genetic circuit of the device secreting Lactoferrin in B.subtilis. This protein has a broad spectrum of anti-infection and anti-inflammatory function.

Reference

1. LUÍS C.S. FERREIRA, RITA C.C. FERREIRA1 and WOLFGANG SCHUMANN. bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors. Anais da academia Brasileira de Ciências, 2005;77(1): 113-124.

2. Morimoto K, Hooper DC, Spitsin S, et al. Pathogenicity of different rabies virus variants inversely correlates with apoptosis and rabies virus glycoprotein expression in infected primary neuron cultures. J Virol, 1999; 73, 510‐8.