Team:NYMU-Taipei/Project/Inhibition/Killing

Introduction

Bee

Defense!!

　　Our main goal is to kill Nosema ceranae without harming the bees.

　　First, we came up with several chemicals that might achieve our goal. They are antimicrobial peptides, such as Apidaecin, [http://parts.igem.org/Part:BBa_K1104300 Abaecin], Hymenoptaecin, [http://parts.igem.org/Part:BBa_K1104301 Defensin1], which are all antimicrobial peptides in bees. Because these chemicals are secreted by bees themselves, they definitely do no harm to bees. Among four kinds of antimicrobial peptides, Abaecin and Defensin are much smaller so it would be easier to synthesize, which was the first reason why we chose [http://parts.igem.org/Part:BBa_K1104300 Abaecin] and [http://parts.igem.org/Part:BBa_K1104301 Defensin1] to treat bees. The second reason was that the sequence of Apidaecin can’t be found now. The third reason is that [http://parts.igem.org/Part:BBa_K1104300 Abaecin] and [http://parts.igem.org/Part:BBa_K1104301 Defensin1] have been relatively well-studied. In addition to antimicrobial peptides, we figured out other chemicals, which are Chitinase, Herein, Protofil, Cytochrome P450. Unfortunately, Chitinase, Herein, Protofil, and Cytochrome P450 have several defects, for example, they are all macromolecules, which make them difficult to be synthesized by E. coli. Moreover, even though E. coli successfully synthesize them, it is too hard to make these chemicals released. According to all of the above, we finally decided to use [http://parts.igem.org/Part:BBa_K1104301 Defensin1] and [http://parts.igem.org/Part:BBa_K1104300 Abaecin] to treat the bees infected by Nosema ceranae.

Background

How Do AntiMicrobial Peptides Work?

　　The cytoplasmic membrane is a frequent target, but peptides may also interfere with DNA and protein synthesis, protein folding, and cell wall synthesis. The initial contact between the peptide and the target organism is electrostatic, as most bacterial surfaces are anionic, or hydrophobic. Their amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by ‘barrel-stave’, ‘carpet’ or ‘toroidal-pore’ mechanisms. Alternately, they may penetrate into the cell to bind intracellular molecules which are crucial to cell living. Intracellular binding models includes inhibition of cell wall synthesis, alteration of the cytoplasmic membrane, activation of autolysin, inhibition of DNA, RNA, and protein synthesis, and inhibition of certain enzymes. One emerging technique for the study of such mechanisms is dual polarisation interferometry. In contrast to many conventional antibiotics these peptides appear to be bactericidal instead of bacteriostatic.

• See more("articles & pictures" modified from) [http://en.wikipedia.org/wiki/Antimicrobial_peptides http://en.wikipedia.org/wiki/Antimicrobial_peptides]

　　After deciding the chemicals that are able to kill Nosema ceranae without harming the bees and how they work, we designed our experiment. The following is our material list as well as experimental procedure.

Circuit Design & Experimental Methods

Circuit Design

[http://parts.igem.org/Part:BBa_K1104300 Abaecin]

[http://parts.igem.org/Part:BBa_K1104301 Defensin1]

Experimental Methods

1. Add Defensin and Abaecin individually into two different test tubes.

2. Add sucrose solution into both test tubes.

(The reason why the above procedures should be done is to lure the bees to take in the sucrose solution with the chemicals.)

3. Split the bees into two groups. One will be fed with the sucrose solution containing any one of antimicrobial peptides daily, which is the experiment group.

The other fed with sucrose solution only is the control group.

4. Make experiment group and control group infected by Nosema ceranae. ( Add ?x10³ spores in the sucrose solution for infecting experiment and control groups, respectively.)

5. Measure the amount of spores in control group daily and plot the replication number of spores against time after inoculation (Day 1~7).

6. Five to seven days after inoculation of the spores is the minimum duration for germination and reproduction of Norsema ceranae.

Therefore, at the time end point, 7 days, all bees are sacrificed by cooling at 4°C and taken out their midgut by forceps.

7. Smash the midgut and observe it under the microscope.

8. Count the numbers of spores in experiment group and control group by RBC counting chamber.

9. Determine the effect of treatment timing of Defensin and Abaecin: Split experimental group into 3 groups.

The first group is fed daily with antimicrobial peptides on day 1~ 3, the second group is fed on day 3~ 5, and the third group is fed on day 5~ 7.

10. Do steps 1–9 three times.