This year’s iGEM team from the University of Toronto has been working on a cell-free method of detecting gold on a paper-based biosensor. Their goal is develop a cheaper, more efficient, and more environmentally friendly method to detect gold for the mining industry. Their detector is GolS, a transcriptional activator that binds gold ions in solution. Following gold ion sequestration, the protein binds to a specific promoter called the P(golTS) promoter region and activates downstream transcription.
IgeM UofT sent us two plasmids of their design. One plasmid (called GolS hereon in, BBa_K2048001) contains the sequence of GolS under regulation of a TetOn promoter and a downstream P(golTS) promoter linked to a LacZɑ sequence, contained in pSB1C3. The other plasmid (called P118A hereon in, BBa_K2048002) contained the same DNA sequences, however the GolS sequence has a point mutation changing a proline to an alanine at position 118. Wild-type GolS in Salmonella enterica is known to bind to other metal ions, such as Cu, but the GolSP118A mutation abolishes specificity for Cu while only slightly decreasing specificity for Au ions1.
One of the goals of our project this year is to synthesize gold nanoparticles. When we found that iGEM UofT was working with a gold ion sensor, we became excited at the possibilities of collaboration. After some brainstorming, we came up with the idea of characterizing GolS in vivo to add onto iGEM UofT’s current data (mainly in vitro, but some in vivo).
We were informed that UofT had performed an analysis of the ability for GolS and GolS_P118A to react to copper ions in solution, using a CPRG assay, which yielded strange results. We thought we would test their constructs for copper and gold binding, as well as silver and aluminum, in order to offer more data to them for better characterization of GolS and GolSP118A.
We initially wanted to determine if our DH5ɑ E.coli would express GolS constitutively, so we set up an preliminary reaction of untransformed cells, cells transformed with GolS, and cells transformed with GolS_P118A, in liquid media with X-gal and different concentrations of gold. We were excited to see that, following day, the cells containing gold and X-gal had blue precipitate, while untransformed cells had no blue product. Due to time constraints and lack of material, we were unable to perform a CPRG or ONPG assay with our liquid cultures. However, iGEM UofT informed us that we could send them pictures of our liquid cultures since they were developing a mobile app that could relate color development to GolS activity. We decided then that our liquid X-gal assay would be acceptable.
We grew untransformed E. coli cells with no antibiotic, E. coli cells transformed with GolS grown in chloramphenicol, and E. coli cells transformed with GolS_P118A grown with chloramphenicol to an OD of 3. We diluted our cells into a final volume of 600 µL of M9 media, having an OD of 1.5. We used Au (III) ions at 1 µM and 5 µM, Cu(II) at 1 µM and 10 µM, Ag (I) ions at 1 µM and 5 µM, and Al (III) ions at 1 µM and 5 µM. Pictures were taken at different time points after the addition of ions, shown in the pictures below. Reactions were incubated at 37℃, 300 RPM. M9 indicates just M9 buffer, M9X indicates M9 buffer with 4.8 µL of 20 mg/mL X-gal. All wells with indicated ions contained M9X.
Throughout the entire process, none of the DH5ɑ cells constructs expressed any blue color, indicating that blue color formation was due to the presence of GolS and P118A. Blue color seems to have appeared in the GolS M9X construct, as well as all ion constructs, possibly indicating that GolS reacted with some ions in the M9 media (which contains calcium and magnesium). However, a darker blue color was observed with the 5 µM Au GolS construct, indicating higher LacZ activity caused by GolS in the presence of Au, as compared to the other ions. This is a confirmation that GolS is performing its intended function of binding to gold ions.
Interestingly, we have found that the GolSP118A construct can clearly bind to gold, copper, and silver ions. Silver ion binding by GolS and has not been documented in the before for GolS, so we have characterized GolS to another degree. Both GolS and GolSP118A do not show any binding affinity for Al ions.
We also wanted to obtain quantitative values from this data, so we attempted to extract the blue insoluble pigment in DMSO. We first spun down the cells (4000 RPM, 5 min), got rid of the supernatant, and lysed the cells with a commercial E. coli lysis buffer. We then added the commercial neutralization buffer, spun the cells again, removed the supernatant, and resuspended the pellet in DMSO. We then obtained absorbance values of our wells.
We found that the solubilized blue pigment had absorbance maximum at 640 nm. As can be seen in the spectrum above, the untransformed DH5alpha incubated with Au ions (which did not have blue pigment) did not absorb at 640nm.
We measured the absorbances of the solubilized blue pigments in each well at 640nm. The absorbances are written under each well. These quantified absorbance values add onto the qualitative data we have generated for our collaboration.
 Ibáñez, M.M., Cerminati, S., Checa, S.K., and Soncini, S.C.. Dissecting the Metal Selectivity of MerR Monovalent Metal Ion Sensors in Salmonella. J. Bacteriol. July 2013 195:13 3084-3092; doi:10.1128/JB.00153-13
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” 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.
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.
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