Part:BBa_K2144003
Coding sequence for Defensin regulated by T7-promoter
Defensin-1
Defensin is an antimicrobial peptide (AMP) that contributes to the innate immune response and is present in both vertebrates and invertebrates. It is highly abundant in tissues involved in the host defence system and its amphipathic character allows it to penetrate and disrupt the integrity of pathogenic microbial membranes for destruction. Honey-bee derived defensin-1 was shown to successfully reduce the viability of biofilm-encapsulated wound pathogens, including P. aeruginosa and our main target, S. aureus [1].
Function
The function of defensin is to lyse the pathogenic cell by permeabilizing the plasma membrane. The surface of defensin has both hydrophobic and hydrophilic regions. Upon binding to the microbial membrane, defensin causes the formation of small channels, disrupting the integrity and increasing permeability of the membrane. This results in efflux (outward movement from cell) of vital components in the cell, thus killing it [2].
Biobrick design
The encoding part of the BioBrick is derived from BBa_K1104301 iGEM13 NYMU-Taipei. The T7 promoter, BBa_K525998 made by the Bielefeld Germany iGEM team 2013, has been inserted to control protein expression under IPTG induction. To regulate the protein expression under T7 promoter control, E. coli containing a T7 polymerase must be used, for instance BL21(DE3).
Characterization
Figure 1. Absorbance measured for different treatments of defensin using S. aureus target.
The graph illustrates how defensin inhibited biofilm-formation in S. aureus. There was a significant difference between the negative control and each of the three volumes of treatment with defensin, thus supporting the role of defensin as a potential combat protein for the inhibition of biofilm formation in S. aureus. However, caution has to be taken with the interpretation since crystal violet also stains bacterial cells and not only biofilm - part of the effect observed may be due to a bacteriolytic activity of defension.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 173
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] Sojka M, Valachova I, Bucekova M, Majtan J. Antibiofilm efficacy of honey and bee-derived defensin-1 on multi-species wound biofilm. J Med Microbiol. April 2016 65: 337-344
[2] Tomas Ganz. Defensins: antimicrobial peptides of innate immunity. Nature Reviews Immunology 3, 710-720 (September 2003)
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