Composite Part

To combat cellulitis, Virginia iGEM designed an innovative solution to produce a device that not only targets the disease-causing bacteria, Staphylococcus aureus, but also permeates the skin barrier to reach the source of the infection: the dermis. To combat the infection, we decided to use nisin A, the primary structure of which is encoded by nisA. In its active form, nisin will attack S. aureus by inducing cell death. To allow nisin to reach the infection and permeate the skin barrier, we decided to use the transdermal peptide, TD-1. A V8 cleavage site was included to ensure the protein is active only in the presence of S. aureus. This resulted in a composite part design that consists of a WELQ cut site for cleavage of epitope tags that may be added on the N-terminal of the peptide, TD-1, a glycine-serine linker, the V8 cut site, and the nisin precursor. We decided to include a linker between TD-1 and NisA to ensure that there are no interactions between the two proteins that would hinder their respective functions. We confirmed the expression of this construct in E. coli BL21(DE3) cells. Specifically, we performed a Western Blot using a whole cell lysate of E. coli BL21(DE3) containing pRSFDuet1-TD1-linker-V8-nisA and concluded that our cells successfully expressed the composite part. Teams can find the composite part in the Registry as BBa_K4853010.

Human Practices

From the beginning of NiSkin, Virgina iGEM prioritized talking to experts and the public to guide our project. When we were determining how to permeate nisin through the skin barrier, we consulted experts including Dr. Stephano Menegatti, Dr. Xin Guo, and Dr. Renquan Ruan to determine which skin-penetrating peptide we should use and whether our project was feasible. After receiving confirmation that it was feasible to attach nisin to TD-1, we wanted to make sure our goals aligned with those of the public and prescribers. Our goals with NiSkin were to have a topical treatment for cellulitis that reaches the dermis, combat antibiotic resistance, and limit the side effects associated with oral antibiotics. From the early stages of the project, we held meetings with dermatologists, infectious disease specialists, and surgeons to gather their perspectives on our goals. From these meetings, not only did we gain information on current treatments for cellulitis and their side effects, but we strengthened our project by adjusting the parts that were suggested. For instance, most of the physicians we consulted had asked about Group A Streptococcus, and so we changed our focus to combatting purulent cellulitis, a subtype of cellulities primarily caused by Staphylococcus aureus.

We also wanted to know what the public thought about the project. To gauge public interest and determine if NiSkin needed any changes to be more attractive to the public, we released a survey (protocol #6001). The responses we received highlighted that people favor topical and oral treatments for skin infections, are extremely concerned about antibiotic resistance, and are highly alarmed about side effects associated with oral treatments. However, these responses were the same reactions that we shared and used to shape NiSkin, meaning that our goals did not need to change. We also introduced our project to the Instituto Tecnológico de Chihuahua, the Dermatology Interest Group Association, and other teams at the Mid-Atlantic Meetup we hosted this year. The questions that we received during these events made us consider how to store our product and NiSkin’s effects on the body.

Throughout the project, Virginia iGEM has consulted with both experts and the public in order to design a product that people are comfortable using and still be effective against cellulitis.

Part Collection

Virginia iGEM designed nine basic parts and three composite parts for a part collection to synthesize their protein of interest, nisin. After consulting the literature, we determined the three essential genes to create modified nisin and made them into basic parts. The nisA gene encodes NisA, which is composed of a leader peptide and the nisin precursor. The nisB gene encodes NisB, a dehydratase that dehydrates serines and threonines in the precursor’s structure. The nisC gene encodes NisC, a cyclase that creates thioether bonds between dehydroamino acids and cysteine thiol groups within the precursor. Both of these post-translational modifications are required to produce nisin with antimicrobial activity. These parts were codon optimized for E. coli BL21(DE3). Furthermore, the nisA gene was altered to include a sequence coding for a V8 protease cleavage site between the leader peptide and the precursor. The V8 site allows for the leader to be cleaved in the presence of Staphylococcus aureus, leaving active nisin. These parts can be found on the iGEM registry as BBa_K4853000, BBa_K4853001, and BBa_K4853002. Four out of five of the remaining basic parts are the primers for the respective genes. BBa_K4853007 and BBa_K4853008 serve as the forward primer and reverse primer for nisA and nisB. BBa_K4853005 and BBa_K4853006 serve as the forward and reverse primer for nisC. Our last basic part is the skin-penetrating peptide, TD1 (BBa_K4853004). We expressed TD-1 linked to NisA within our composite parts.

Each of the composite parts encodes NisA and TD-1 but varies in which features they have. Part BBa_K4853012 encodes a protein with TD1 and a V8 site between the truncated leader of NisA and the nisin precursor. Part BBa_K4853009 encodes a protein that includes only a V8 site between TD-1 and NisA. The final composite part, part BBa_K4853010, encodes a protein that includes a linker sequence followed by the V8 site in between TD-1 and NisA. When one of these composite parts are inserted into the same chassis containing the basic parts for nisB and nisC, it will not only produce nisin, but nisin that is fused with TD-1 and has the potential to permeate the skin barrier.