Team:Northwestern

SCIN - Self-regenerating Chitin INduction

Chitin, found in the exoskeletons of insects and crustaceans, is one of the most abudant substances in nature. Like keratin in skin, it comprises the protective outer layer of these animals. Our chitin expression platform involves generating a layer of chitin from a lawn of bacteria in response to an external molecular cue. This cue induces chitin synthesis (fast) and cell lysis (slow). This system allows for a build-up of chitin followed by cell lysis and subsequent release into the top layer of the lawn. Abrasions expose cells to the external cue for self-repair. In this way, we create a regenerative chitin biolayer with potential medical and industrial applications.

Project Description: Bacterial Skin

Our main project is to simulate mammalian skin using chitin in E. Coli. This process involves four major components: Lawn Formation, Chitin Synthesis, Bacterial Apoptosis, and lac-Operon Signaling.

A thick bacterial lawn is generated by plating ydgG knock-out mutants (acquired from the Keio Collection) in an enriched agar plate. The protein product of ydgG plays an integral role in AI-2 transport. ydgG knock-outs exhibit increased motility which should ultimately allow for a thick and level lawn.

The 3.5kbp Chitin Synthase III gene is extracted from the Saccharomyces Cerevisiae genome. Chitin Synthase III catalyzes the polymerization of chitin by transferring UDP-N-acetyl-D-glucosamine to an N(1,4 N-Acetyl-beta-D-glucosaminyl) to produce N+1(1,4 N-Acetyl-beta-D-glucosaminyl).

Apoptosis of cells is achieved by using the bacteriophage lysis cassette built by the Brown '08 iGEM Team. The cassette includes Holin, Endolysin, and Rz Protein genes. These enzymes puncture and degrade the cell membrane which results in lysing of the cell and the release of synthesized chitin.

Once the lawn is established, an IPTG solution is sprayed over the lawn to induce chitin synthesis (fast response) and apoptosis (slow response). IPTG mimics allolactose which binds to the LacI repressor, causing LacI to detach from DNA and allowing RNA Polymerase to begin transcription. This will allow for a build-up in chitin followed by apoptosis and subsequent release into the extracellular environment. Thus, any abrasions to the chitinous surface can be repaired by spraying IPTG.

In mammalian skin, mitosis occurs in the basal layer of the epithelial cells and cells travel outwards towards the surface of the skin as they mature. As the cells move further away from the basal layer, they begin to die due to lack of nutrients. As they die, their cytoplasm is released and the cells are filled with keratin, thus forming a continuously regenerating protective layer on the outer-most part of epithelial layer. Our project is modeled after this. The bacterial lawn produces chitin only at the top-most layer and these cells then undergo apoptosis to result in the formation of a chitinous layer at the surface of the lawn. The critical difference is that the bacterial colony is not internally controlled (controlled by IPTG spray), but this is something that can be remedied and a possible goal for future teams.

SIGNIFICANCE OF CHITIN:

Medicinal Use:

• Wound and burn treatment/healing
• Hemostasis for orthopedic treatment of broken bones
• Viscoelastic solutions for ophthamology and orthopedic surgery
• Abdominal adhesion treatment
• Antibacterial and antifungal agents
• Tumor therapies
• Microsurgery and neurosurgery
• Treatment of chronic wounds, ulcers and bleeding (chitin powder)

Industrial Use:

• Food/Pharmaceutical/Agricultural/Cosmetic thickener, stabilizer
• Water resistant properties
• Dietary supplement
• Water purification
• Biodegradable
• Edible microcrystalline films used to preserve food
• Sequestering of particles (i.e. oil)

Northwestern University

Northwestern University in Evanston, IL, combines innovative teaching and pioneering research in a highly collaborative environment that transcends traditional academic boundaries.

Our lab facilities are located at: Technological Institute 2145 Sheridan Road, Evanston, IL 60208

Find out more about [http://www.northwestern.edu Northwestern University]

Find out more about [http://www.mccormick.northwestern.edu/ McCormick School of Engineering]

Find out more about [http://www.weinberg.northwestern.edu/ Weinberg College of Arts and Sciences]

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