AMP. coli

To achieve our goal we incorporated antimicrobial peptides (AMPs) into our medical dressing. AMPs, are stable peptide that have extensive ability in bactericidal effects. Unlike antibiotics, AMPs can puncture the cell membrane to kill the bacteria therefore bypassing bacterial antibiotic drug resistance mechanisms. [1] Besides, the peptides also have ability to help skin recovered. [2] After reading numerous of research articles, we selected two kinds of AMPs: Signiferin and Epinecidin-1 as our reagents.

Signiferin is a peptide came from the skin mucus of Crinia signifera. It demonstrated effectiveness in killing Methicillin-Resistant Staphylococcus aureus (MRSA), and has already been demonstrated by the TU-Delft 2013 iGEM team. [3] Epinecidin-1 is a peptide came from the skin mucus of Epinephelus coioides. It has ability to help wound healing and has been proven by animal studies, and was selected as an additional reagent. [4] By combining these two properties, we believe that can develop a wound dressing that may be useful in trauma patients without the additional risk of developing drug-resistance.

To control the AMPs expression and secretion, the Lac operon was used and treated signal peptide into our system. Helping peptides secret into culture medium. [5, 6] After purification of the peptide we will be testing the effectiveness of our synthetic AMPs. We will test macro-dilution of MRSA and in vitro wound healing assay for epithelial cells line (HaCaT) and micro vascular endothelial cells (HMEC-1). Out goal is to create a wound dressing that is effective in inhibiting bacterial growth and assisting wound healing process.

• Epinecidin-1:

1.	From the skin mucus of Epinephelus coioides a kind of fish.
2.	Has function of killing bacteria.
3.	In addition, it has the ability to help wounds healing and has been proven by animal studies.

• Signiferin:

1.	From the skin mucus of Crinia signifera a kind of tree frog.
2.	Have function of killing bacteria.
3.	Have great ability in disinfect Methicillin-Resistant Staphylococcus aureus (MRSA).
4.	Had already been kindly proved by the 2013 TU-Delft iGEM team.

1.	Helps AMPs to secret out of E. coli.
2.	From Streptomyces lividans to transport chitinase C to secretion system, which has been proven to work in E.coli by reference.

Based on AMPs to develop into a potential material of wound dressing.

References

[1]	Yeaman, M.R. and N.Y. Yount, Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev, 2003. 55(1): p. 27-55.

[2]	Lai, Y. and R.L. Gallo, AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol, 2009. 30(3): p. 131-41.

[3]	TU_Delf, i.t. 2013; Available from: https://2013.igem.org/Team:TU-Delft.

[4]	Huang, H.N., et al., Use of the antimicrobial peptide Epinecidin-1 to protect against MRSA infection in mice with skin injuries. Biomaterials, 2013. 34(38): p. 10319-27.

[5]	Tokuyasu, K., et al., Production of a recombinant chitin deacetylase in the culture medium of Escherichia coli cells. FEBS Lett, 1999. 458(1): p. 23-6.

[6]	Fujii, T. and K. Miyashita, Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans. J Gen Microbiol, 1993. 139(4): p. 677-86.

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