Team:Concordia/Description
Why did we choose this project?
This year, our iGEM Concordia 2016 team has chosen to use a combination of synthetic biology and engineering technologies to design our project, ‘Combat Cells: League of Enhanced Microgladiators’. The major goal of our project has been to generate our own tiny armored gladiators by equipping different cell types with various ‘nano-weapons’, which are nanoparticles of our own creation, and placing these little personalized warriors on a microfluidics chip so that they can undergo battle. Before these microgladiators reach the final battle ‘arena’, they are subjected to a series of treacherous conditions that challenge the robustness of the mini-warriors. In other words, these armored cells are subjected to different conditions, similar to an obstacle course - pushing these Combat Cells to their ultimate limits. There are several reasons as to why we chose this particular project…
(1) Our main drive was our desire to create something fresh, new and exciting that could harmonize the appeal of entertainment value with the essential core element of iGEM: synthetic biology. By generating a fusion between science and media, we were looking to design something with a fun twist would also encompass some novel technologies. Ultimately, we wished to prepare a project that had never been done before. With the rising interest in micro-entertainment, and the increasing use and appeal of new microfluidics technologies, we wanted to merge these two areas. Knowing that many iGEM teams had not yet ventured into the use of microfluidics to control cells for entertainment purposes, we decided to steer our project toward this path, and that is how we came to create ‘Combat Cells’.
(2) We wanted our project to be able to reach a greater audience. While most scientifically-oriented research projects are not as communicable to a laymen audience, we aimed to devise something that would be more understandable for the public. In this, we could gain the attention of many, and also educate them about synthetic biology and engineering technologies in an amusing environment. We hoped that this would bring an unconventional feature to scientific education. This is also why we chose to become involved in human practices activities, to bring our project to the public and get them involved in this new science. We used this opportunity to also gauge the comprehensibility of our experimental process and receive feedback from different members of the general community. We tried as much as possible to integrate the public’s ideas into our experimental design, so that not only our team would enjoy our project, but so would our target audience: the whole community. Moreover, we really aspired to get people inspired about synthetic biology and science as a whole. By making the project entertainment-oriented, we believed that this would be a good way to get people excited about microfluidic technologies and synthetic biology. Entertainment through media is very relatable to the current population, and to further this clause we designed both an easily accessible app as well as a mini-game to grab the attention or a larger audience through various means that might answer to the assorted media preferences of our audience.
(3) While many research projects are only really presentable through demonstration or description, our goal was to take this aspect one step further. Our hope was to make a project that our audience could become involved in, so that they could get a real feel for the power of synthetic biology in versatility - science is not only for basic analysis and characterization, but can be used in many other areas, including entertainment, which may not always be obvious. We knew that the "Combat Cells" idea could really garner an interactive and integrative element to it. To truly respond to this portion of the project’s design, we structured our project accordingly, with special consideration for the obstacle course/battle-dome microfluidics chip and the way in which the cells’ armor could be personally tailored. Ultimately, we wanted to allow people to form their own "Combat Cells" teams and come up with their own individualized "Combat Cells". Essentially, people would form teams who would choose a strain and type of cell, select which "nano-weapons" they would want to equip their cells warriors with, pick the conditions they would want their gladiators to experience through the obstacle course, and have their cells battle other teams. In essence, teams would battle their customized cells in the controlled environment of the microfluidics chip "battle-dome," similar to the larger-scale robotics-style "Robot Wars".
(4) Despite the core part of our project revolving around science in entertainment as a way to get people involved and to educate the public, we also really hope to be able to improve upon and innovate pre-existing technologies. We wanted to expand on their potential applications of microfluidic technologies which is an ever-growing area of engineering, with consideration for cellular control and testing the versatility of different chip conditions for cell subjection purposes. Further, we wanted to be able to optimize pre-existing plant-based nanoparticle or ‘nano-weapon’ synthesis methods which are more eco-friendly as opposed to the more common chemical-based methods. Next, we were interested in improving upon existing nanoparticle-cell-surface attachment techniques, without cell internalization of nanoparticles - which is not often done in current research. On the molecular biology side of things, we wanted to optimize an existing method of combined nanoparticle synthesis and cell-surface attachment, to speed up the process. In this we could facilitate two major steps of our project, which could be used for other research projects.
Nanoparticle Synthesis
To generate nanoparticles, we are harnessing the reductive powers of plants such as garlic, aloe vera and cabbage. Plants possess a variety of biomolecules that are capable of reducing and stabilizing metal ions to form nanoparticles. The sizes and shapes of nanoparticles can be manipulated by varying the amounts of plant extract and metallic solutions used for synthesis. Using a Transmission Electron Microscope (TEM), the nanoparticles synthesized can further be characterized. In this we aim to develop optimized methods for controlling the shapes and sizes of the nanoparticles using plant-mediated synthesis. Furthermore, incorporating this eco-friendly and cost-effective approach to synthesizing nanoparticles will allow our project to reduce the amount of waste we may produce during nanoparticle synthesis.
Beyond the use of plants, we are also using chemical methods to synthesize nanoparticles. The Martin and the Turkevich methods are common chemical nanoparticle synthesis. Martin uses the strong reducing power of sodium borohydride to reduce gold nuclei into small gold nanoparticles in the 3-6nm diameter range. The gold solution that is being reduced, also contains trisodium citrate whose ions will cap and stabilise the gold nanoparticles. The Turkevich method uses the moderate reducer and strong stabiliser trisodium citrate whose reducing power is increased by heating the solution containing silver nuclei. This reduction of silver nuclei would lead to formation of nanoparticles in the 40-60nm diameter range.
Most importantly is our recombinant method. According to literature we have found (Tsai et al.), we can force E. coli cells to make nanoparticles for us. The protein MelA is a tyrosinase that oxidizes L-DOPA, and through a set of downstream reactions, melanin and eumelanin (a type of melanin pigment) are formed. Eumelanin, in the presence of gold ions, can create gold nanoparticles in the range of 5-15 nm (Tsai et al.).
We aimed to improve the characterization of MelA for Gold, which was input into the BioBrick registry by iGEM Cambridge in 2009 (BBa_K274001), by exploring the ability for MelA to use tyrosine as a substrate instead of L-DOPA. We wanted to determine if eumelanin could be formed from tyrosine, and from there we wanted to optimize nanoparticle formation through the use of tyrosine. We constructed a part containing the sequence for MelA under the regulation of a constitutive promoter. This construct would continuously produce MelA and would be used to determine cytotoxicity of melanin and eumelanin production. Results of our experiment using tyrosine can be found by clicking here.
Once we determined tyrosine's ability to be used a substrate, we would then use it to form gold nanoparticles. Our E. coli cells would also be transformed with a plasmid containing the sequence for a fusion protein of FhuA and a gold-binding peptide (GBP). FhuA is a transmembrane protein found endogenously in E. coli, and GBP is a peptide sequence capable of binding to gold nanoparticles based on charge interactions. Thus, this recombinant method would involve synthesizing nanoparticles and attaching them to the surface of cells.
Click here for detailed protocols
PLANT BASED SYNTHESIS METHODS:
CHEMICAL SYNTHESIS METHODS:
Recombinant Method and Parts Employed
The recombinant method will involve the expression of the MelA gene, which will lead to the formation of eumelanin. This protein will in turn reduce the gold nuclei within E. coli cells and will intracellularly form gold nanoparticles. FhuA-GBP, a transmembrane protein linked to a gold-binding peptide, will be expressed in order to induce extracellular display of gold nanoparticles.
RECOMBINANT METHOD:
HOW DO WE KNOW IF NANOPARTICLES WERE FORMED?
Nanoparticle Attachment
The next step following nanoparticle synthesis is nanoparticle-cell attachment. To do this, our team’s goal is to develop an effective linkage method between the nanoparticles and the cell’s surface, using both model organisms Escherichia coli and Saccharomyces cerevisiae. One of our methods includes the creation of a gold “Nanoshell” made from gold nanoparticles coated with L-cysteine surrounding the outer surface of yeast cells. Another one of our methods involves involves coating silver nanoparticles with polyelectrolytes and attaching these to the surface of both yeast and E. coli cells. We intend to study the relationship between nanoparticle abundance as well as localization on the protective qualities offered to the cell by nanoparticle cell coating.
NANOPARTICLE ATTACHMENT:
Microfluidic devices dominate everyday life but are not apparent, for example: laser printers, pregnancy tests, pH paper, and medical diagnostics1.
Microfluidics is an interdisciplinary branch of science that studies the behavior of fluids through microchannels and the technology of manufacturing micro-devices containing chambers and tunnels where liquids are confined to. Downscaling lab-based analysis systems has created a whole new class of devices; providing a platform to develop a lab-on-a-chip. Microfluidic devices give the advantage of manipulating and controlling fluids in the range of micro- to pico-liters small. This implies that analytical processes such as: sampling, sample treatment, reaction, detection and data analysis are downsized to the micrometer-scale. Smaller analysis platforms means low reagents, multiple assays (done simultaneously), and high resolution. Microfluidics, therefore, is an attractive platform to integrate simple micro-sized system operations that apply to a whole lab.
There are many different microfluidic platform technologies being used that have many applications such as: microchannels (droplets), microchannels (continuous), paper microfluidics, digital microfluidics, and slip-chips2. No matter what type of microfluidic paradigm being used the fundamental basis remains the same; fluids are directed, mixed, or separated through these microchannels.
Microfluidics exploit the physical and chemical properties of fluids. There a three important parameters to keep in mind when fluids enter a microchannel density, ρ, pressure, P, and viscosity, η. Microfluidics systems require shear stress (application of external forces) to be applied to a fluid in order for the system to properly operate. The fluid flow conditions are due to the relationship between viscosity and inertial forces acting on the fluid system. In a microfluidic system fluid flow is categorized into laminar and turbulent flow.
Laminar flow3 is defined as layers of liquid that flow in a uniform fashion, these layers do not mix with neighboring layers (Reynolds < <1). Turbulent flow is defined as fluid particles that move in irregular paths(Reynolds >>1).
Because of their accessibility and the precision with which these handheld devices are able to handle liquids, they have emerged as an important new paradigm in synthetic biology. These devices have revolutionary applications in high-throughput screening, biological and chemical assays, single cell analysis and automation of wet bench work. Our project focuses on droplet microfluidics, a channel-based, two phase system. In this system, monodispersed sub-microliter aqueous droplets are formed in a surrounding oil phase. By flowing a cell suspension as the aqueous phase of this system we are able to form distinct droplets containing cells. By optimizing the flow rate and the concentration of cells in the cell suspension, we are able to predictably generate droplets which contain only a single cell. The ability of these devices to isolate a single cell from a cell suspension is what will be taken advantage of in order to clash two cells against each other in combat.
[1,2,3] Shih, Steve. Microfluidic Devices. Digital image. Https://users.encs.concordia.ca. N.p., n.d. Web. 17 Oct. 2016. <https://users.encs.concordia.ca/~sshih/doc/Lecture%201_X.pdf>.
References
Chandran, S. P., Chaudhary, M., Pasricha, R., Ahmad, A., & Sastry, M. (2006). Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotechnol. Prog., 22(2), 577-583.
Konnova, S. A., Danilushkina, A. A., Fakhrullina, G. I., Akhatova, F. S., Badrutdinov, A. R., & Fakhrullin, R. F. (2015). Silver nanoparticle-coated “cyborg” microorganisms: rapid assembly of polymer-stabilised nanoparticles on microbial cells. RSC Adv., 5(18), 13530-13537. Retrieved from http://pubs.rsc.org/en/content/articlehtml/2015/ra/c4ra15857a
Nan, J., Xiao-Yu, Y., Guo-Liang, Y., Ling, S., Jing, L., Wei, G., Ling-Jun, D., et al.“Self-repairing”nanoshell for cell protection. Orr, J. C., & al., E. (2005). Supplementary information. Nature, 437(7059), 681-686.
Rastogi, L., & Arunachalam, J. (2013). Green synthesis route for the size controlled synthesis of biocompatible gold nanoparticles using aqueous extract of garlic (allium sativum). Advanced Materials Letters.
Tamileswari, R., Nisha, M. H., & Jesurani, S. S. (2015). Green Synthesis of Silver Nanoparticles using Brassica Oleracea (Cauliflower) and Brassica Oleracea Capitata (Cabbage) and the Analysis of Antimicrobial Activity, 4(04), 1071-1074.
Tsai, Y.-J., Ouyang, C.-Y., Ma, S.-Y., Tsai, D.-Y., Tseng, H.-W., & Yeh, Y.-C. (2014). Biosynthesis and display of diverse metal nanoparticles by recombinant Escherichia coli. RSC Adv., 4, 58717-58719. Royal Society of Chemistry. Retrieved from http://xlink.rsc.org/?DOI=C4RA12805B
