Team:Concordia/Demonstrate
WORKS IN REAL WORLD CONDITIONS
The main goal of our iGEM Concordia 2016 team has been to design and create a miniature battle-dome to have armored cells undergo a controlled duel. More specifically we aimed to generate nanoparticle ‘mini-weapons,’ attach these to varying cell types’ surfaces, then have these armored cells battle in a controlled environment in our personally-designed microfluidics chip ‘battle-dome’. In order to reach that goal, many mini-goals needed to be accomplished along the way. While most of these mini-goals were successfully reached, our overall goal of finally battling our cells was not. Due to both time constraints and speed bumps along the way, we did not have the chance to combine each of our mini-goals to attain that final step. Although we did not complete the entirety of our overall goal, each of the parts that were achieved were highly significant, in that they leave that final step of the cell battle open to future testing. If given a bit more time, we would have been able to wrap up what we started. Given the time constraints, we accomplished much more than we had initially anticipated, and are proud to say that we attained the majority of our goals. Whilst we met success in most of our experimental endeavours, we did encounter some road-blocks, as to be expected with any type of research. Both our major successful and unsuccessful results are described in the following text, to provide you with the overall storyline of our experimental process.
The first step toward our target was the synthesis of a variety of nanoparticles with unique properties using a multitude of techniques. This was how we would be able to compare the effectiveness between use of nanoparticles with distinguishable sizes, shapes and compositions, as ‘weapons’ for our cellular gladiators in damaginged their cellular opponents. This would ideally also serve, in the future, to allow cell teams to be customized in accordance with ‘nano-weapon’ preference. In this, we were also able to consider the ideas of the public when deciding which nanoparticle synthesis methods to use to generate our ‘mini-weapons’ based on which shapes and compositions they believed would be most successful in serving as a ‘weapon’ - a useful element in our human practices efforts. To get that diversity of our ‘nano-weapons,’ we used several researched pre-existing nanoparticle synthesis methods. Two of these methods are the commonly used chemical techniques: Martin method and Turkevich method. In order to demonstrate our awareness toward environmental health and safety, we also performed three plant-based synthesis methods using aloe vera extract, cabbage extract, and garlic extract. Each and every one of these synthesis methods were successfully accomplished, providing us with a wealth of options for our nanoparticle ‘weapon’ choices. All these results can be found here
Our next goal was to attach these nanoparticles to the surfaces of cells so they could be used as a type of “weapon” during the cell battle. To accomplish this, we used several attachment methods. The nanoshell method of attachment creates a gold nanoshell around yeast cells that we hope will serve as a form of protection for the cells. We successfully attached gold nanoparticles to yeast cells using this method. Nanoparticles synthesized from the Martin method of synthesis and nanoparticles purchased from Cedarlane were both successfully attached to these cells. Attachment of gold nanoparticles synthesized using garlic extract was also attempted but it was not as successful as the Martin or bought nanoparticles. Nanoparticles synthesized using aloe vera extract were also successfully attached to yeast cells. In addition to this, we managed to attach nanoparticles from Cedarlane to temperature sensitive CDC28 mutant yeast cells that do not grow at 37°C. All these results can be found here
The Cyborg method of attachment was used to attach polyallylamine coated silver nanoparticles to yeast cells. We successfully attached silver nanoparticles synthesized using the Turkevich method to the surface of yeast cells. Silver nanoparticles that we made using cabbage extract were not able to attach to yeast cells. The Cyborg method was supposed to work for attachment to both yeast and E.coli cells. However, all attempts at attaching to bacterial cells were unsuccessful and resulted in the cells aggregating or dying. It was also a challenge to view E.coli samples under a microscope because they are significantly smaller than yeast cells.
To test how effective the nanoshell was at protecting the yeast, we attempted several trial of a lyticase test. Following the nanoshell attachment procedure, lyticase would be added nanoparticle coated cells and control cells. Unfortunately the test did not seem to work because both the nanoparticle coated cells and control cells had a similar drop in OD. More details on this test and our results can be found here
Another way that we had hoped to test the protective abilities of our nanoparticles was by exposing our nanoparticle-coated cells to an exotoxin. One of the teams we collaborated with was NAWI-CRAZ, they sent us a plasmid containing an exotoxin that could be secreted into liquid media. However, we were unable to transform the plasmid into DH5a cells so we were unsuccessful at testing our nanoparticles’ ability to protect the cells from the toxin.
The recombinant method that we had intended to use to synthesize nanoparticles and attach them to E.coli cells was unsuccessful. However, we successfully completed several steps that would allow us to use this method in the future. We fused the ferrichrome iron outer membrane transporter (FhuA) with a gold-binding protein (GBP). This could be used for a gold-binding method because GBP would bind gold nanoparticles while the FhuA would display these nanoparticles on the cell membrane of E.coli cells. However, when we attempted to express FhuA-GBP in E.coli using an expressible plasmid, we were not successful. Another success of ours was placing MelA, a gene that produces melanin, under a constitutive promotor. We also managed to create a temperature-sensitive CDC28 mutant by knocking out the wild-type gene using CRISPR. More details on this can be found in our recombinant results page
The final phase of our project consisted of engaging our nano-particle coated cells into a duel. This battle would take place on a microfluidic chip, which we designed. This allowed for the battle to take place within a confined microscopic environment and it also allowed us to manipulate the direction the cells moved in. In this phase, we wanted to test the strength of nanoparticles as either weapons or shields against one another. This would be analogous to a battle and would ultimately provide entertainment value for the educational purpose of our project.The production of a functional microfluidic chip was constructed in three phases: design the chip on AutoCAD, fabricating the master chip, and then fabrication of the microfluidic chips through photolithography. As a result, fully functional microfluidic chips were produced. A major obstacle in producing a microfluidics chip was designing a chip that would support and function properly with isolated cells. It was also important that it result in a collision of two droplets containing single cells--this concept has never been attempted before. Optimization of microfluidic chips require patience and trial and error; a main issue concerning the microfluidic chip was simulating a violent collision of the two merging droplets with isolated cells.Therefore, electrodes were inserted in order to induce a turbulent flow, this would facilitate the simulation of cells battling each other. In order to optimize flow rate, we flowed blue and orange dyes through different inlets and saw them mix within the microfluidic chip. This allowed us to optimize the flow of liquid through two different channels which later merge into a single channel. Following this, we wanted to flow cell suspensions through our chip. We were hoping to isolate single cells within a droplet, however, we ran out of time and could not fully optimize our system to do so. Despite having not generated the results we had entirely hoped for, our accomplishments have provided enough of the groundwork for subsequent Concordian iGEM teams to progress the Combat Cells legacy, the ultimate fun and engaging learning tool for educating and inspiring potential scientists to pursue a role in the field of synthetic biology. All these results can be found here
WORKS IN REAL WORLD CONDITIONS: TESTING STRESSORS
In order to determine whether the 'Combat Cells' on our microfluidics chip 'battle-dome' would be able to withstand the application of a variety of added stress conditions, imitating an obstacle course-styled setup, cell viability tests were done. To verify the viability of cells, or detect the damage done to cells under the stress conditions applied, propidium iodide (PI) dye was used. PI is a DNA intercalating agent, that absorbs light at a wavelength of 535 nm, and when bound to DNA, fluoresces at a wavelength of 617 nm. Since cellular damage would most likely translate as cell puncture or membrane breakdown, thus release of DNA, PI is a useful agent in detecting cell death or damage; once a cell is damaged, the dye can access the cell's DNA and fluoresces. Thus PI was used as a read out for a stress condition affecting cell integrity. Several conditions were tested including: low pH, high pH, surfactant addition, osmotic stress, high salt, detergent addition, and mechanical stress. We were able to test two uncoated cell types, both Escherichia coli and Saccharomyces cerevisiae. The next step would be to run these tests on other cell type strains and on cells coated with a variety of different nanoparticles, attached using numerous attachment methods. The information collected during this testing provides us with the control data, which would be used to compare with data to be collected for nanoparticle-coated or armored cells. The goal here was to use this test information and be able to apply the different stress conditions tested onto the microfluidic chip as the Combat Cells are passing through the device. This would eventually be used to test the limits of their nanoparticle armor in defending or protecting the cell against harsh cellular environments. For now, this information tells us which conditions are worth testing on nanoparticle-coated cells, given how uncoated cells react.
The first step of the experimental procedure was procuring cell cultures of Escherichia coli and Saccharomyces cerevisiae. The cells in the culture were grown in regular growth medium, then were re-suspended in PBS (phosphate buffered saline). A solution of PI (2 mg/mL) re-suspended in PBS was used for addition to cell samples. Each solution of stressor was added to cells in PBS at a ratio of 1:1. For instance, 75 uL of E. coli in PBS was added to 75 uL of 0.001M HCl acid, yielding an overall concentration of 0.0005M HCl in solution. As soon as the cell solution is mixed with the acid solution, PI is added to the mix at 1:100 (thus 1.5 uL added to the total solution volume of 150 uL). As this is done, a UV-VIS spectrophotometer is used to measure the fluorescence over 30 second time intervals for a total of 9 minutes (as of the addition of PI into cell solution) to verify changes in sample fluorescence. An increase in sample fluorescence indicates cell damage has occurred. This was similarly done for each of the conditions. Specifically for the mechanical stress test, the cell-PBS mixture was subjected to gold nanoparticle collisions by vortexing the mixed cell-nanoparticle solution for 45 seconds prior to addition of PI and UV-VIS reading. For each condition, control samples were tested where a 1:1 solution of PBS (without cells) and the stress solution had PI added and the UV-VIS read.
With the data collected on fluorescence intensity, graphs were plotted to display the information for both cell types under each stress condition, for both the experimental and control samples. A trendline is used to characterize each of the trends for E. coli and S. cerevisiae cell damage under each condition. Also, a control trendline is provided for comparison against each of the cell damage trends. An equation in each trendline's respective color is used to indicate the slope of the trend. A positive or increasing slope indicates that the cell is undergoing damage (fluorescence increases), while a negative/decreasing or plateau slope indicates that the cell is not being significantly damaged (no change in fluorescence or a decrease).
Using the information from each of the graphs, two histograms were created to describe the timing at which the stressor begins to affect the cells in question, and also to determine at which point in time the cells are most affected by the respective stressor. In some cases, there is no cell damage or significant damage inflicted upon the cells with a given condition, thus a line or '-' is used to indicate that the data is not significant.
In terms of the actual data collected, several trends are worth describing. In the case of low pH conditions, it appears as though E. coli is damaged by the acidic environment progressively over time. As for S. cerevisiae at low pH, there is also damage that occurs, but quicker for a brief time early on, once subject to the HCl. This tells us that low pH is a significant stressor for both cell types that could be adjusted in intensity and used to test nanoparticle armor efficiency. Next, high pH conditions for E. coli showed slow increasing cell damage over time, while for S. cerevisiae the impact of a base cause significantly more cell harm than for bacteria over time. Like for low pH, it would be worth verifying the impact of high pH on nanoparticle-coated cells. Under surfactant conditions or PBST with mild 0.1% Tween detergent, both bacteria and yeast cell types did not show any form of damage in terms of PI fluorescence. In other words, this stressor was sustained by the cells and would not be worth testing against armored cells unless the condition was perhaps intensified by using a higher detergent Tween concentration. Following, osmotic stress was applied to the cells, where doubly distilled water was added to solution. Under this given condition, neither E. coli nor S. cerevisiae showed significant cell damage response. This provides that the osmotic pressure was not enough for the cells to be harmed, making this condition unworthy of further testing on armored cells. The next condition tested was a high salt stressor, using 1.5M sodium acetate (NaAc). In this case, the E. coli showed a plateau of fluorescence for PI indicating no change in cell integrity upon addition of the stressor. Instead, for S. cerevisiae, the salt appeared to significantly cause change in cell integrity, as PI fluorescence increased very slightly over time with most prominence. This means that only nanoparticle-coated yeast would be worth testing under high salt conditions.
Next, strong detergent conditions were verified using 0.5% sodium-dodecyl sulfate (SDS). As anticipated, both cell types were damaged providing that the detergent is something that should be tested against armored cells. The damage inflicted upon yeast was quicker than that upon the bacteria which was slower and took more time to occur. Finally, the last condition tested was mechanical stress inflicted upon collision of gold nanoparticles with cells. Contrary to what was expected, both cell types were not harmed by the mechanical stress. This is considerable as this infers about whether small spherical gold 'nano-weapons' may be less effective in causing cell harm versus other shapes, which could also be tested.
Further each of these conclusions provide us with an idea of the cell's limits and capabilities with respect to resisting specific stress levels and types of stress. Also, the histograms provide times at which cellular damage is inflicted significantly under a respective condition. This is information that could guide us to determine how long we subject our cells to each condition in the microfluidics chip. It is worth mentioning that the PI to cell concentration ratio was optimized for these tests by measuring the fluorescence of a series of cell concentrations prepared as cell-PBS solution dilutions. This information was plotted in two graphs for each cell type. These cells were homogenized to expose their DNA and test the limit of fluorescence saturation dependent on cell concentration. In the case of E. coli, where the absorbance was measured at 600 nm, the undiluted cells had an absorbance of 0.5282. Undiluted quantities of cells were used for each cell stressor situation tested. For S. cerevisiae, the absorbance was also measured at 600 nm, but the undiluted cells had an absorbance of 1.6582. Given such a high absorbance, the PI signal became saturated, therefore a 5-fold dilution of cells was used for yeast. 5-fold diluted quantities of cells were used for each cell stressor situation tested.
