Team:Concordia/Collaborations
NAWI Graz iGEM Team
We were excited to find another team working on simulating cell to cell battles in the iGEM competition. NAWI-Graz’s project, titled “The Last Colinator, (https://2016.igem.org/Team:NAWI-Graz)” focuses on using two strains of E. coli, each containing a unique toxin-antitoxin system, for the purposes of creating a battle.
Although we weren’t using toxins and antitoxins in our own battle system, we still aimed to collaborate with NAWI-Graz on two fronts. We wanted to have a discussion about the ethical dilemmas in our projects; after all, both our teams are forcing cells to fight for instructional and entertainment purposes. Additionally, we wanted to find a way to utilize their toxin-antitoxin system in our own project.
Ethics Discussion:
On September 19th, 2016, we had our first meeting with iGEM NAWI-Graz over Skype. We talked about our projects and our experiences (both the good and bad!). We then got on the topic of the ethics of our project. In our research, and in much of the domain of molecular biology, model organisms such as E. coli and S. cerevisiae are manipulated for the purpose of furthering human understanding of the molecular world. These cells, although microscopic, are considered living entities, a part of the tree of life which also includes humans, plants, and much more.
Knowing this, we asked questions related to the rightness or wrongness of our projects. In a macroscopic world, it is seen as deplorable to force living animals to fight for human entertainment, such as dog or cock fights. Yet, in the microscopic world, we humans don’t have any issues with forcing cells to fight one another, or to die in the case of untransformed DH5α E. coli cells on LB+Amp plates. For what reason or reasons do we not care about manipulating our microscopic brethren to die for us? Is it because of size differences? Is it because there are vastly more single-cell organisms than humans? Because single celled organisms do not have nerve cells meaning they can’t feel death? Is the alternative just too problematic that the ends justify the means?
During our Skype call, we asked these questions and more to members of iGEM NAWI-Graz’s team. We touched on the aspects of cell manipulation, cloning, stem cell developments, and all sorts of hot topic ethical issues in science. Much like us, they had asked members of their school’s community how they felt about a project manipulating cells. And much like us, most members of academic institutions did not have issues with our projects, going as far as calling them cool and innovative. However, asking members of the general public seemed to arise some worries, relating our goals to potential bioterrorism and genetic mutation.
Both of our teams came to an agreement that a grave problem lies in the media, the middleman between science and the common public. The way scientific endeavours are presented by the media and received by the public can help or hinder the trust that people have in any subfield related to biology. iGEM NAWI-Graz’s “The Last Colinator” has potential for future studies in new metabolic pathways and alternatives to antibiotic use. iGEM Concordia’s “Combat Cells” has potential for future studies in bio-nanotechnology and single-cell-to-single-cell interactions on microfluidic chips. Yet, for people without an educational background in biology, the first thought goes to germ warfare, leading to superbugs and a threat to the human race. Even though our team made clear to the public that there are dangers associated with working with nanoparticles (as we’ve addressed in our SOP, LINK HERE), we had to emphasize to people that our work was controlled and disposed of carefully, and there is no chance that our work dangers people outside of the laboratory. For iGEM NAWI-Graz, although their toxin-antitoxin system is a heritable trait, they emphasized that the work is only performed in a lab strain of E. coli that cannot grow outside the lab.
The enlightening ethical discussion with iGEM NAWI-Graz helped Concordia’s iGEM team form a better understanding of topics that biologists do not always care enough to address during their work. It also helped us to shape our SOP in a manner that, we hope, anyone can clearly approach our project in a safe manner.
Above: Lukas from iGEM team NAWI-Graz in Austria, with Jason, Thiban, Farhat, and Alaa from iGEM team Concordia in the corner.
Experimental Design
Another goal we had set out for our collaboration was to somehow incorporate NAWI-Graz’s toxin-antitoxin battle system into our nanoparticle-coated-cell battle system. After some team brainstorming, we came up with an idea to characterize the efficiency of our nanoparticles protecting E. coli from toxin invasion.
Here is a general layout of the experimental design:
NAWI-Graz had sent us their “Blue plasmid” construct. This plasmid contains the gene coding for a blue chromoprotein, constitutively expressed. It also contains the sequences for toxin ccdB and antitoxin ccdA. CcdB, which inhibits DNA gyrase, is known to be secreted in the media, while ccdA remains in the cell. The plasmids also contains the sequence for DAM-Methylase, which NAWI-Graz uses in their project as a mutation-inducer for their purposes.
The first step of the plan was to transform the DNA into DH5ɑ E. coli cells, followed by a colony PCR to confirm the plasmid presence. The production of the toxin and the antitoxin are induced using IPTG, and the toxin would be secreted in the growth media. Theoretically, this toxin could be isolated from the cells through centrifugation, as they would stay in the growth media while the cells would be pelleted. This media containing the toxin would be used for a different type of experiment, as shown below.
Our intention was to determine whether or not nanoparticle-coated cells would protect against a toxin.We hypothesized that our nanoparticle-coated cells would offer some sort of protection of cells from the toxin. We based this hypothesis on the idea that our silver-coated E. coli cells would have altered abilities to pump external substances into the cell. Our nanoparticles, which are citrate-capped and coated and neutralized using a cationic polymer, are evenly distributed in a non-selective manner along the surface of E. coli cells.
This experiment would be performed by monitoring the difference in optical density over time of our coated cells in contact with toxin. The necessary controls would be uncoated E. coli cells in contact with the toxin (to confirm toxin activity), and both coated and uncoated E. coli without toxin.
Unfortunately, we could not complete this experiment, as we had difficulty transforming the Blue Plasmid into E. coli. We likely diluted the received DNA in too much water since the measured DNA concentration was below the linear range of our spectrophotometer, leading to multiple failed transformations. Another possible reason for low transformation is that the toxin is under the regulation of a lac promoter and operator, which have some leaky expression. If the antitoxin is not expressed while some toxin is expressed, this may be killing the host cell, which does not endogenously produce either ccdA or ccdB.
Survey for iGEM TecCEM
The iGEM TecCEM 2016 team reached out to iGEM Concordia to ask if we were interested in collaborating with them by completing their survey. The two main focuses of the survey were self-medication and nosocomial infections. Members of iGEM Concordia completed the survey and gained some useful knowledge by doing so. We learned that a nosocomial infection is a microbial infection gained through medical equipment. For example,such an infection can be caused by the bacteria Acinetobacter baumannii. TecCEM iGEM 2016 is working on a project that aims to prevent proliferation of Acinetobacter baumannii. Link to the survey
