Team:Utah State/Project

Since there is such a wide diversity of AMPs in nature, we decided to select our peptides from eight different species. The map below shows all of the organisms that we decided to draw from.

Figure shows the diversity of AMPs used by Utah State iGEM team 2013

EcAMP-1 (BBa_K1162001)

EcAMP-1 is derived from the grass species Echinochloa crus-galli. It consists of 37 amino acids and is helical in structure, held together by two disulfide bonds. This feature enables a unique hairpin structure which is not found in most other AMPs. EcAMP-1 has shown to have a toxic affect against a variety of fungi and protists. The mode of action for EcAMP-1 against fungi appears to be the prevention of hyphae elongation (Nolde et al., 2011).

Spheniscin-2 (BBa_K1162002)

Spheniscin-2 is a potent antimicrobial peptide whose DNA sequence of the king penguin. Studies have shown that in nature the King Penguin secrete Spheniscin-2 into its stomach to preserve food for long periods of time by preventing bacterial decomposition of the food. In the wild the male usually incubates the egg for the last 3 months before the egg hatches. In general the female will then come and feed her young shortly after. However, if the mother is delayed the survival of the young can still be insured by the male (Thouzeau et al 2003). However, storing food for long periods of time in a warm stomach would lead one to assume that bacterial degradation would be a limiting factor. However, studies have shown that Spheniscin-2 has a wide range of activity against many gram positive, gram negative, and even fungal microbes, which allows the male King Penguin to store food for long periods of time (2.). Thus, there is a wide range of use associated with Spheniscin-2. The potential of interweaving Spheniscin-2 with spider silk holds many promising results.

Grammistin-Pp1 (BBa_K1162003)

Grammistin-Pp1 is an AMP found in skin secretions from the clown grouper fish and is made up of thirteen amino acid residues. This AMP is used primarily to fend off predators. Although initially thought to have little antimicrobial properties, studies proved that grammistin Pp1 target cell membranes by interacting with phospholipids. Grammistin Pp1 has been shown to be lethal against many gram-negative and gram-positive bacteria including B. subtilis and S. aureus. A primary reason for our selection of grammistin Pp1 was the high concentration needed to inhibit the growth of E. coli. (Yokota et al., 2001)

CG-Defh1 (BBa_K1162004)

CG-Defh1 originates from immune cells in an oyster species. As a defensin AMP, CG-Defh1 contains four disulfide bonds and is most effective against gram-positive bacteria. The specific mode of action for this AMP is to inhibit peptidoglycan synthesis in the bacterial cell wall. This makes CG-Defh1 unique from many AMPs in that it does not target cell membranes. In fact, it was the first defensin from an invertebrate to exhibit antibacterial properties. Most importantly, CG-Defh1 inhibits the growth of S. aureus, a dangerous cause of nosocomial infections in the world.

Scygonadin (BBa_K1162005)

Scygonadin is produced in a species of mangrove crab to prevent bacterial invasion. It is the primary defense against dangerous microbes that thrive in marine environments. Scygonadin has been shown to be effective in inhibiting both gram-positive and gram-negative bacteria including M. leteus, S. aureus, C. glutamicum, and A. hydrophila. This AMP has been previously studied for its production via recombinant DNA in E. coli using a pTrc-CKS vector and a C-terminus 6x His-tag. No studies were done with a 10x His tag or spider silk inserts. (Peng et al., 2010)

LL-37 (BBa_K1162006)

LL-37 is an antimicrobial peptide (AMP) from the cathelicidin family of antimicrobial peptides, isolated from the Human (Homo sapiens). It is a 37-residue helical peptide found throughout the human body, exhibiting a broad spectrum of antimicrobial activity as well as a variety of immunomodulating effects (Dürr, Ulrich H.N., et. al 2006). It has the distinction of being the first and only cathelicidin member discovered from humans, and is extremely well studied and characterized for this reason. The peptide is produced via epithelial cells as well as leukocytes in places such as the skin, gastrointestinal tract, and the respiratory tract. This AMP is is cleaved from a larger protein, hCAP-18, in a series of modifications to yield its active form (Sorensen, O.E, et. al 2001).

WAM-1 (BBa_K1162007)

An antimicrobial peptide from the cathelicidin family of peptides, isolated from the tammar wallaby (Macropus eugenii). WAM-1 is one of several identified peptides that are produced in the mammary gland throughout lactation in order to supply protection from infection to the young wallabies that have underdeveloped immune systems. This is evident through the down-regulation of these peptides in the tammar wallaby milk at around 100 days after birth, the same period that the wallaby young develop immunocompetence (Wang, et. al 2011). Similar to other members of the cathelicidin family, the WAM-1 antimicrobial peptide has been shown to be effective against a broad range of bacterial pathogens (Kastin, A. J. 2013). Specifically, a group of researchers from Australia (Wang, et. al 2011) have tested this peptide, isolated from its natural source, against several gram-negative (E. coli, P. aeruginosa, among others) and gram-positive (B. subtilis, S. aureus, among others) bacterium with minimum inhibitory concentration (MIC) values that indicate highly effective antibiotic activity. To our knowledge, this antimicrobial peptide has not been expressed and purified recombinantly in E. coli.

OH-CATH(3-34) (BBa_K1162008)

OH-CATH(3-34) is an antimicrobial peptide isolated and characterized from the king cobra (Ophiophagus hannah)(Zhao et al 2008). A member of the cathelicidin family, OH-CATH Cathelicidins in general have been well characterized and are known to have a diverse C-terminal antimicrobial domain connected to a conserved cathelin-like N-terminal domain (Nizet et al 2003). Specifically, OH-CATH has been studied for its toxicity against a wide range of gram negative and gram positive bacteria. OH-CATH has also been characterized for its potent activity against multi-drug resistant bacterial strains including MRSA (Li et al 2012). Further studies by (Zhang et al 2010) have shown that removing the first few amino acid residues of the peptide dramatically reduced the hemolytic activity while having only minute effects on antimicrobial properties. This attribute would be highly desirable in any usage that would interact with humans and helped to determine our choice our peptides. The combination of high bacterial toxicity with low hemolytic activity justify more testing into the precise mechanisms of OH-CATH and its ability to be used as a scaffold for other antibiotic purposes.

There are many problems with producing AMPs on a large scale. Natural purification of these AMPs is a slow process and is generally inefficient. A more viable option is chemical synthesis. However, this process is extremely costly (Li, 2011). Finding a way to overcome these problems is key to the development of a new antimicrobial product. Producing AMPs in bacteria such as E. coli using recombinant DNA techniques could provide an answer to increasing production rates and decreasing costs.

The first obstacle faced in producing AMPs in E. coli is how to avoid inhibiting E. coli growth itself before satisfactory AMP concentrations have been achieved. E. coli is easy to engineer, is quick to grow, and inexpensive to use. This makes it the most attractive bacteria to use for protein production. To prevent the culture from dying before producing the desired AMP, strong transcriptional promoters must be included to reduce gene expression to a very low basal level (Sorensen & Mortensen, 2004). This promoter allows the E. coli culture to reach stationary growth phase before being induced to begin high-level gene expression of the AMP. In this and most studies, isopropyl-β-D-thiogalactopyranoside (IPTG) is used as an inducer.

We have chosen our antimicrobial peptides because they belong to several families of proteins and have the ability to inhibit a broad range of pathogenic microorganisms.

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 (with RFC 23 standards) in pSB1C3 an E. coli as the chassis. AMP amino acid sequences were first back translated to obtain DNA sequences. In all cases a methionine (atg) had to be added. These DNA sequences were then codon optimized for expression in E. coli using the Life Technologies GeneArt ® software program. Sequences were verified not to contain any illegal restriction sites (EcoRI, XbaI, SpeI,and PstI). AMPs were designed with RFC 23 under consideration to allow for protein fusions. In this project protein fusions were important for AMPs to be purified with 10x His Tag, GFP, and also to allow AMPs to be fused to spider silk.