Team:Penn/Nissle

We have seen how the complex interplay between public opinion and science innovation can drastically affect the adoption and success of a new technology, such as our team's bacterial drug delivery system. As pointed out earlier, pervading public opinion towards a bacterial therapeutic system such as the one developed by our team this year would most likely be negative. In an effort to address some of these issues, we have set out to investigate ways our system could be made more palatable to the general public.

One recent concept that we have identified as gaining general acceptance within the general public is the incorporation of "probiotic" organisms into a daily diet. Many foods, such as yogurts now advertise the presence of "probiotic" bacteria and there are "probiotic" supplements containing live bacterial cultures as well. One particular probiotic, E. Coli Nissle 1917 has attracted attention not only from the public, but also from the scientific community, where its potential beneficial properties have been investigated. The Nissle strain is notable for its lack of virulence factors and decreased immunogenecity [1]. These traits are what make Nissle a popular probiotic. Nissle has also been found to preferentially colonize tumors, proliferating wildly in the borders between live and necrotic tissue, a highly desirable trait for any potential cancer treatment [2]. Additional investigation has demonstrated that intravenously administered Nissle exhibits a similar behavior in breast cancer mouse models, and expression of recombinant azurin prevented cancer metastasis in mice [4]. However, the therapeutic potential of Nissle is not limited to cancer treatment. Nissle is also capable of enhancing wound healing through recombinant expression of human epidermal Growth Factor in the epithelial linings of the body, as well as reducing modulating responses to allergens [5,6].

Based on these properties, we believe that demonstrating that our drug delivery system can be implemented in Nissle 1917 would be the first step to addressing the potential hesitance that the public may have to bacterial based therapies. Because the chassis for our system is a probiotic, we can avoid not only the technical difficulties of ensuring that the host for our system is inherently safe, but also proactively address (or at least minimize) the initial "knee-jerk" reactions that many members of the general public may have to the idea of a bacterial therapeutic. Furthermore, In order to fully realize the potential of Nissle, it is important to be able to easily and consistently manipulate and change its genetic information. Therefore, we have produced and characterized a process for generating chemically competent Nissle 1917 that can be produced in any standard microbiology lab, allowing future iGEM teams to unlock Nissle 1917's full potential.

As shown below, chemically competent Nissle 1917 bacteria produced by the lab was capable of taking up the pDawn-ClyA light-based cytolysis module of our system. Furthermore, these bacteria can begin to express genes encoded on the plasmid, such as the kanR gene, which confers resistance to the Kanamycin found in the LB plates.

As shown below, chemically competent Nissle 1917 bacteria produced by the lab was capable of taking up the pDawn-mCherry plasmid modeled on the pDawn-ClyA light-based cytolysis module of our system. Furthermore, these bacteria exhibit blue light-dependent expression of mCherry, a red fluorescent protein, as seen on the right of the photo.

1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C overnight.
2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).
3. Shake vigorously at 37 °C to OD600 ~0.25-0.3.
4. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice.
5. Centrifuge the cells for 10 min at 5000 g at 4°C.
6. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.
7. Centrifuge the cells as above.
8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol.
9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.
   • Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.