Difference between revisions of "Team:Michigan/Gold Integrated"

We sought the opinions of experts in the field to consult on our project and design during the initial drafting period. The original design ambition was to create a temperature controlled system for a mini-bioreactor, increasing the availability of basic biochemical treatments to resource-strapped communities. However, after consulting with our faculty advisors, we realized that our project would not address the underlying problems that plague those communities, and that the practical limitations of the design would severely limit its impact. Instead, they suggested that the design carried the potential of a temperature-controlled kill switch. See our Silver Human Practices Page for our detailed thought process.

After designing our kill switch, we decided it would be best to gain some input from someone who had worked with pathogenic strains of bacteria. We met with Andrew Lowell, a postdoctoral fellow at the Sherman Lab at the University of Michigan Life Sciences Institute. Prior to our meeting, our killswitch was only envisioned as being used in pathogenic lab strains. However, Dr. Lowell brought to our attention the problems that the escape of non-pathogenic antibiotic resistant bacteria cause. By incorporating our switch into common lab strains such as DH5 Alpha E. Coli, we could prevent the horizontal transfer of antibiotic resistance to other potentially harmful strains in the environment. Also, our initial design called for two plasmids, one with the TlpA36 promoter expressing a lambda phage repressor that held a lysozyme on the other plasmid in check. After consulting Dr. Lowel, this design was scrapped before we began cloning it because it was too impractical to have two plasmids. Eventually we settled on the design explained in depth on the Project Description page. Following this meeting, we were able to brainstorm additional potential applications of a temperature-dependent kill switch.
They were as follows:

-Our killswitch could be incorporated into pathogenic strains, like MRSA. This safety precaution could limit the risk of working with such strains, and thus lower the biosafety level needed. This would enable more labs to study antibiotic resistant pathogenic strains.

-Plan our design based so it may be incorporated into both pathogenic and non-pathogenic strains.

-We discussed an alternative to the holin endolysin system, a temperature induced auxotroph as a kill switch mechanism.

We then decided it would be ideal to talk to a geneticists and acquire knowledge on details we should consider for our kill switch as well as come up with additional applications. Dr. Csankovszki is a genetics professor at the University of Michigan who studies dosage compensation in C.elegans. She was intrigued by the idea of a temperature-dependent kill switch and was able to think of several applications during the meeting.
Ideas:

-Consider the secondary effects the repressor proteins could have on the cell, if any. (While we did not get to this step it would be a great point to build on for other teams and further projects).

-Use our kill switch in school teaching labs as an added safety precaution so research in classes can be more extensive without worrying about biosafety levels.

-Use our kill switch as an alternative to sonicators when lysing cells to obtain proteins.

One of the main reasons we decided on designing a kill switch was the public’s rising concern with GMOs. We believed that our switch could potentially aid in alleviating the public’s fears, and begin establishing a dialogue between the public and experts, discussing the possible benefits and pitfalls of GMO use. The panelists’ discussion also helped us understand the benefits of our project from the general public’s perspective.
This is what we learned:

-According to Dr. Fisher, many customers are wary about GMO, and fear the uncertainty associated with them. Many companies profit from including GMO-free labels on their products.

-The media has the tendency to exaggerate the risks associated with GMOs, thereby forcing customers to question the safety of GMO products. GMO classification further complicates the issue as the label obscures what is thought of as ‘natural’ and ‘unnatural’ practices.

-GMO products (both foods and GMO products for other applications) can be dangerous depending on the context. For example, GMO crops can harm the environment, if herbicide resistance genes are passed on to weeds. Our internal “kill-switch” can, therefore, serve to decrease the negative stigma associated with GMO products, by ensuring containment of GMOs to the laboratory (or any other environment intended for the GMOs).

During the creation of the panel, we drafted questions and discussed them with the four panelists. Their input helped inform our approach to designing experiments to evaluate the risks of our modification. Discussion of public fears about synthetic biology informed future experiments that we planned. As mentioned in this document (link to essay that is connected to silver HP), public concerns about synthetic biology often lead to a label of artificial and unnatural. We planned a series of experiments to demonstrate that cells carrying our construct in a plasmid or as an episomal integration do not behave differently from the standard lab strain of Dh5 alpha cells. A representative environmental setting such as a countertop or soil could be sterilized using physical or chemical techniques. After that, our cells or Dh5 alpha cells could be spread onto the surface and grown at 37 degrees Celsius. After 3 days, the surface would be swabbed and bacteria on the swab would be streaked onto a plate. To test the effects of the bacteria on other organisms in the environment, microbial diversity could be measured using PCR amplification of 16S rRNA genes.

After talking to Dr. Csankovszki, our natural next-step was to present our findings to a biochemist, namely, Dr. Stockbridge. Dr. Stockbridge studies mechanisms and structures of membrane transport proteins in bacteria, and praised our project as clever, utilizing a flexible mechanism that could have diverse functions.
This is what we learned from her:

-Dr. Stockbridge thought the idea of using the system to lyse the cells when purifying proteins was very clever and useful.

-A future project idea was that of the “conditional auxotroph.” Production of amino acids would be dependent on environmental temperature. Cells would be able to synthesize an amino acid at lower temperatures but not at higher temperatures. Then, cells can be labeled with the addition of modified amino acids, such as p-fluorophenylalanine. This would solve a problem common to biophysicists and biochemists when analyzing proteins with NMR.

-Another application was to incorporate a mutant ion channel without a ligand binding site, which would cause the cell to burst due to osmotic shock.

-Finally, a suggestion made in order to have tighter control of the system would be to use genetic regulatory elements instead of protein folding.

The incorporation of our killswitch into pathogenic strains, which could lower the BSL2 certification needed to work with pathogenic strains such as MRSA to BSL1, was pointed out as a potential application. This would enable more labs to study antibiotic resistant strains. The idea of using our temperature controlled kill-switch design to repress the biosynthesis of an essential cellular building block, like alanine, was also proposed as a more reliable and specific way to kill bacteria upon exposure to low temperature. Another suggestion was that our killswitch would be used to induce the lysis of bacteria during protein purification, bypassing the need for expensive sonication equipment.