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 batte 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
