Team:Utah State/Project

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Antimicrobial peptides (AMPs) are known for their ability to kill bacteria that are harmful to its host and are part of the innate immune system of most multicellular organisms (Andreu & Rivas, 1998). AMPs from various organisms have shown the ability to inhibit many types of gram-positive/gram-negative bacteria, protists, fungi, and other organisms. The outstanding diversity of AMPs found in all organisms on the planet contribute to a constantly growing list of peptides with varying properties. The size, amino acid sequence, charge, conformation, hydrophobicity, and amphipathicity all contribute to the activity of an AMP. Most AMPs contain fewer than 100 amino acids and are positively charged (Giuliani et al., 2007).

Organisms everywhere produce AMPs that allow them to thrive in their distinct environments. For example, Crocodiles contain AMPs in their blood, which allow them to engage in territorial struggles without subjecting themselves to infections brought on by bacteria living in the murky waters that they call home. King Penguins produce unique AMPs in their stomachs to allow for extended food storing periods to feed their young. Marsupials have also shown to be unique hosts of AMPs. The Tammar Wallaby relies on the production of AMPs for prolonged lactation of its young, which remain in the mothers pouch for 9-10 months (Wang et al., 2011).

The diversity of AMPs is likely another example of adaptations occurring in organisms due to environmental pressures. It has been determined that most AMPs are derived from precursor sequences that are quite similar. Conserved regions on AMPs even between different species of organisms has led to this belief (Zasloff, 2002). The changes present in the current AMP sequence are probably due to the thriving of organisms containing mutations in the AMP sequence that allowed them to gain an advantage over other individuals lacking the mutation (Zasloff, 2002). These AMPs have been tested over many generations in their host organisms and makes them fascinating for studies in which their activity is tested against a variety of organisms.

With so many AMPs, it is no wonder that a variety of mechanisms are possible for bacterial cell killing (Epand et al., 1999). Most AMPs that have been studied follow the Shai-Matsuzaki-Huang (SMH) model (Zasloff, 2002) (Lai & Gallo, 2009). This model overviews the interaction of a peptide with the outer layer of a cell membrane. The general flow of this model includes the “carpeting” of the membrane with the AMP followed by the displacement of lipids in the membrane, and finally the lysing of the membrane. The SMH model can be seen below.






Antimicrobial Spider Silk

Antimicrobial spider silk proteins (functionalized spider silk) could be used to make antimicrobial spider silk fibers or films. A previous study by Gomes et al. 2011 demonstrated that it is feasible to produce antimicrobial spider silk films. The Utah State iGEM team in 2012 was able to successfully produce spider silk proteins in E. coli with the use of BioBricks and successfully spin spider silk protein that was approximately 25.4 kDa in size. The parts that were submitted to the registry by Utah State in 2012 are RFC 23 compatible which allows for protein fusions, hence the addition of RFC 23 compatible AMPs to spider silk protein at the C or N terminal would be highly desirable.

The spider silk generator (BBa_K844016) created by Utah State in 2012 was able to produce proteins of 25.4 kDa. An increase of spider silk subunit repeats at the genetic level would enable a larger (and therefore ‘stronger’ fiber) to be produced. This year we proposed to double the number of spider silk repeat units to 8 and attach AMPs to either the C or N terminal ends of the spider silk. The purification of this antimicrobial functionalized silk would be performed with a 10x His-Tag fused to the spider silk, at the opposite terminus as the AMP. The possibility of "masking" the properties of the AMP were taken into account with our genetic design, which is why we did not create this fusion protein with the AMP located in between the spider silk and 10x His-Tag proteins. The figures below demonstrate the design of antimicrobial functionalized spider silk.



All constructs were assembled in pSB1C3 an E. coli as the chassis. AMPs were designed with RFC 23 under consideration to allow for protein fusions.

Sílvia C. Gomes, Isabel B. Leonor, João F. Mano, Rui L. Reis, David L. Kaplan, Antimicrobial functionalized genetically engineered spider silk, Biomaterials, Volume 32, Issue 18, June 2011, Pages 4255-4266