Future Work

1.For vaccine question, glycoprotein was expressed in B.subtilis. Our final goal is to activate the immunity systems of dogs through this vaccine. We will inoculate mice the vaccine in the future for test. Meanwhile, two kinds of expression systems can be used for B.subtilis as antigen vector. We will try to display glycoprotein in the surface of Spore of B.subtilis through Chromosomal-encoded way in the future.

2.For moving question, we want to know whether the modified bacteria can be vomited by flea. We confirmed to iGEM Safety Committee that:

2.1 We only work on the Bacillus part of our project for the iGEM competetion 2013 and shall theoretically demonstrate our ideas for the flea part but will NOT raise or release any live fleas for this competetion.

2.2 We will NOT work on live fleas until we have addressed the biological safety and environmental concerns stated in our communication.

In the future, we shall consult and collaborate with a reputable research group to use clean lab grown fleas, certified to be free from pathogens.

In the following we provide the method we found in some papers to finish this experiment. Meanwhile for rearing flea, please see the section of Method for rearing flea of our project.

1.To prove Bacillus subtilis can live in the proventriculus of flea, we designed the following procedure:

i.Express GFP in Bacillus subtilis.

ii.Put Bacillus subtilis into blood and feed fleas with the following device (shown in Figure 1)

Fig. 1. shows the device of rearing flea.

iii.Observe fleas using a fluorescence microscope.

2.To prove the flea will vomit Bacillus subtilis:

i.Express GFP in Bacillus subtilis.

ii.Put Bacillus subtilis into blood and feed flea with the following device (shown in Figure2).

Fig. 2. shows the device of rearing flea.

iii.Put fleas into the device without Bacillus subtilis; after the fleas are fed, check the blood to see if there is Bacillus subtilis in the blood.

iv.Repeat step iii.

Fig 3. To prove the flea will vomit Bacillus subtilis

3.For safety question, the two antimicrobial peptides were successfully expressed in B.subtilis. We found all of our antimicrobial peptides can kill gram-positive bacteria. Staphyloccocus aureus is found in the internal environment of flea. Maybe the antimicrobial peptides can make flea free of Staphyloccocus aureus. For gram-negative bacteria, we found that the subject of 2011 iGEM team FATIH TURKEY is to kill gram negative bacteria. They succeeded in using Limulus anti-lipopolysaccharide(anti-LPS) factor to stop the growth of gram negative bacteria. They chassis is B.subtilis as well.

In the future, we will try to combine Antimicrobial peptides with anti-LPS to develop multi-functional antimicrobial peptides which can kill different bacteria.


[1] M. Vobis,J. D'Haese,H. Mehlhorn,el. Experimental quantification of the feline leukaemia virus in the cat flea (Ctenocephalides felis) and its faeces[J]. Parasitology Research, 2005, 97 Suppl 1: S102-6.

[2] B. J. Hinnebusch,M. L. Rosso,T. G. Schwan,el. High-frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut[J]. Molecular Microbiology, 2002, 46(2): 349-354.