Team:Harvard/Design


Design: Modifying E.Coli to Reach Our Goal

The crux of our project was to genetically modify E.Coli to secrete keratinases. In order to design and develop a prototype, we accomplished the following:

  1. Identified keratinases that are non pathogenic and have synergistic kertinolytic activity to efficiently degrade targeted keratin atop the skin
  2. Proved that our E.coli could successfully secrete the keratinases extracellularly by utilizing the curli fiber secretion pathway and a pBbB8k plasmid backbone
  3. Successfully suspended our modified E.coli cells with desired keratinases and media in alginate gel, proving viability and ability to change pore size of alginate suspension.

1) The Enzymes:

  • Our first goal was to secrete three keratinases from Onygena Corvina: endoprotease 6877, exoprotease 8025, and Metallopeptidase (oligoprotease). These enzymes were identified by Lange, L., Huang, Y., & Busk, P. K. (2016)1 who denoted a possible synergistic effect of the three keratinases in completely degrading keratin. After secreting the three enzymes, we would be able to test how effectively these enzymes were able to degrade keratin.
  • One way to test enzymatic degradation of keratin is to use a Keratin Azure assay. First, a piece of sheep’s wool (containing keratin) is impregnated with a blue dye (keratin azure) in a high pH medium and overlaid on a keratin-free basal medium2. Keratinase production is measured by the release and diffusion of the keratin dye into the medium below. The Keratin Azure assay lends to a cost effective means on initially testing for keratinase activity. Further, we'd like to test the keratinolytic activity of the enzymes on the skin itself. Unable to do so, suitable alternatives to using human skin while also emulating said environment are testing on pig skin and on human established cell culture lines.

2) Plasmid Backbone design:

  • Image of pBbB8k vector generated by SnapGene

To insert the sequence of our desired keratinases, we decided to replace the CsgA of E.coli and ligate the gene using Gibson Assembly. The addition of secretion tags to our keratinases would enable them to be recognized by the curli pathway mechanism, allowing them to be secreted extracellularly (particularly across the CsgG secretion channel). Additionally, we decided to switch the promoter of our systems from IPTG to Arabinose because the potential toxicity of IPTG render it unfit for clinical use on human skin.

3) The Alginate Suspension:

  • The vision of our project was to place the modified E.Coli in an alginate patch which would allow the bacteria to continuously secrete the keratinases as treatment for the skin. This particular element is important because one of the current obstacles facing patients and doctors alike is compliance; continued secretion and ability to adjust how much keratinase is produces and released are therein key features of our prototype. Therefore, after successfully engineering our E.Coli to secrete the endoprotease, exoprotease, and oligoprotease, we then wanted to test the feasibility of suspending our cells in alginate.
  • Since the patch would be in contact with the human skin, we wanted to choose a gel that is biocompatible and has pore sizes that can easily be altered. Alginate displays both of these properties and exhibits mild gelling which would allow us to control the environment in the suspension, allowing only the keratinases to secrete out of the gel (leaving the E.Coli remaining in the alginate), and preventing other microorganisms from entering the patch. Below, two options for creating these gels are discussed.

    • Option 1

    First, we explored how to encapsulate cells using microfluidics. In this case, the size of the pores and cell-containing alginate beads are determined by the concentration of our alginate cell solution and the volume ratio of CaCl2 (divalent cross-linking solution) to hydrated alginate. This protocol, developed by Linas Mazutis,* Remigijus Vasiliauskas, and David A. Weitz, produces alginate hydrogel particles that are pancaked shape and should contain only a few bacterial cells.3

    Microdevice used here was 10 micrometer deep, 20 micrometer wide → encapsulated particles are ≅ 5 micrometers thick, and ≅16 micrometers wide

    Option 2

    In addition to using microfluidics, we also created a protocol with the help of the Langer lab to more easily suspend cells in alginate. That protocol can be found under the "Protocols" tab of our wiki.

    Synopsis

    • By completing these experiments, we hope to not only achieve the goals we set for ourselves in design, but also set the groundwork for other researches to use our project as a springboard for future studies of keratinases and their applications.
    • We found that there was a lack of scientific literature addressing the possible uses of different keratinases, whether in industry, medicine, or other fields of research.
    • It was also difficult to find a method of inexpensively synthesizes the three keratinases in lieu of cloning.
    • We hope that modifying E.Coli to produce these keratinases rapidly by arabinose induction offers a more accessible way for researchers to study and discover more uses of these class of enzymes

    Sources

    1. "Microbial decomposition of keratin in nature-a new hypothesis of ...." 12 Jan. 2016, https://www.ncbi.nlm.nih.gov/pubmed/26754820. Accessed 17 Oct. 2018.
    2. "(PDF) Determination of keratin degradation by fungi using keratin azure." 1 Aug. 2018, https://www.researchgate.net/publication/8427180_Determination_of_keratin_degradation_by_fungi_using_keratin_azure. Accessed 17 Oct. 2018.
    3. "Microfluidic Production of Alginate Hydrogel Particles for Antibody ...." 21 Jul. 2015, https://www.ncbi.nlm.nih.gov/pubmed/26198619. Accessed 17 Oct. 2018.