Safe and Inexpensive Approaches to Advance Synthetic Biology
TL;DR: We have two projects! One is antibiotic free plasmid retention, the other is a cheap PCR machine!
Abstract: Alternative methods of plasmid maintenance and PCR amplification accelerate the construction of new biodesigns, reduce cost, and avoid environmental hazards. Plasmids are typically maintained in cells by encoding enzymes that hydrolyze or otherwise detoxify antibiotics added to the medium. However, this process carries an inherent risk for spreading antibiotic resistance to native bacterial populations through lateral gene transfer. The Hok-Sok toxin-antitoxin system, a natural internal maintenance cassette relying on internal mRNA silencing, presents an alternative to common antibiotic-based methods since it does not rely on exogenous drugs. We are also developing an integrated, microcontrolled thermocycler using common household components. Using nichrome wire and a motorized fan for air circulation, the programmable prototype is an inexpensive, versatile thermocycler or plate incubator. Because the material and construction costs are a fraction of dedicated instruments, the newly developed unit will find broad application among nascent synthetic biologists in underfunded environments.
Hok/Sok
Antibiotic resistance is a necessary selection factor for transgenic bacteria using plasmids as vectors. This staple of genetic engineering has been met with opposition with valid claims that the addition of antibiotics to the environment harms native species and poses a risk to unwanted antibiotic resistance through lateral gene transfer.
The Hok/Sok system has naturally evolved in bacteria as a means of plasmid retention, and is capable of addressing the issue by providing a selection factor for plasmid retention without the dangers of antibiotics and risk of lateral gene transfer. The Hok (host killing) gene codes for a mRNA which lies dormant in its initial secondary structure. As it is degraded by exonuclease, it assumes a translatable secondary structure which produces an apoptosis triggering protein. The Sok (suppression of killing) gene codes for a mRNA transcript that binds to the Hok mRNA, preventing it from being translated. The complex is eventually degraded by nuclease. Hok has a half life of 20 minutes, while Sok has a half life of 30 seconds. As long as both genes are present, the cell remains alive. After cell division, should the cell not retain the plasmid of interest which contains Hok/Sok, Hok mRNA remains the cytoplasm for 20 minutes, while remaining Sok is degraded. Since the cell does not contain a Sok gene, no Sok is being produced to save the cell from being killed by Hok. This system is very similar to current antibiotic resistance systems, only without the necessity for antibiotics themselves, resolving the issue of environmentally safe plasmid retention.
ELI5:
When scientists change the DNA of bacteria, the bacteria don't like it and want to go back to normal. To force the bacteria to stay changed, scientists add antibiotics (the same ones you take when you're sick). Adding a lot of antibiotics can cause problems, like other bacteria getting sick and the bad DNA spreading (which we don't want). To make sure the bacteria stay changed WITHOUT using antibiotics, we developed Hok/Sok. It works the same as antibiotics does. If the bacteria tries to go back to normal, it dies. If the bacteria stays changed, it lives. The only difference between this system and antibiotics is 0% antibiotics are used.
Polymerase Chain Reaction or PCR is a common tool used in the field of biology to amplify DNA or RNA. Invented by Dr. Kary Mullis, PCR is conducted trough cycling DNA, primers and enzyme through various temperatures. Generally starting with a value near and above 90 degrees Celsius; used to break the Hydrogen bonds between double strands a process called denaturation. The machine then cools down to annealing temperature, with values near 50-60 degrees, at this point primers are able to attach to the template strand of DNA. This stage is then followed by extension temperature, around 72 degrees, at this point the polymerase is able to extend and add nucleotides to the primer.
Although the process of amplifying genetic material is remarkable, the hardware needed to do it is relatively simple-- all that is required are three different temperatures which are maintained by the machine, enabling the enzymes and template to do the work of PCR. Current PCR machines cost thousands of dollars, and although there exists open source, DIY PCR machines, their costs still range in the hundreds of dollars. Here at the University of Maryland, we thought that that was an absurd notion. PCR, because of its simplicity and utility, is a robust tool for the diagnosis of many diseases both in the developed and developing world. Making the device cheaper would give more people accessibility to this platform. Accessibility enables further innovation and development of novel methods for disease detection and this in turn enables better and faster diagnosis and treatment both in the developed and developing world.
Another major advantage of "cheap" is education. Here at the University of Maryland, we acknowledge that iGEM is a competition, however we also understand that this competition is also a collaboration. It is an opportunity for all of us to learn from one another and serves as the foundation for future discovery, innovation, and new projects. We hope that our work with the PCR machine will inspire many more teams to tackle designing hardware. We hope that our current collaborations with Duke University foster better and more innovative projects from both of our teams. And most important, we hope that our efforts will be able to inspire the future generation of iGEMer's and the newest members of the iGEM community; high school students.
I remember, along with my fellow teammates, learning about PCR by cutting up little paper nucleotides and putting them into a brown bag and then having our hands act as the "polymerase" that would pluck the nucleotides out and match them with the template strand we were given. I remember taking away very little from this "lab" other than a few paper cuts. In subsequent years, I went through a few internship programs where I was able to learn in greater detail the steps of PCR, eventually learning how to design primers, program the machine, and setup my own reactions. However, I believe that if we truly want to bring synthetic biology to the public, we have to allow them the opportunity to actually do PCR, not through a paper bag which is conceptual understanding, but a real reaction where the end products are the real deal, actual amplified DNA. We still have a ways to go... the enzymes have to become cheaper pipettes need to become cheaper, but designing a below 50 dollar PCR machine is the first step in this endeavor.
CHIP1 is our first design and employs, in many respects, a more conventional PCR design. CHIP1 utilizes two peltier units below an aluminium heating block to heat the PCR tubes sitting inside the block. We use a temperature sensor to detect the temperature of the wells in which the PCR tubes are housed. The sensor then reports back to the Arduino unit, which regulates the energy flow to the peltier units, thereby heating and cooling the block and the tubes.
CHIP2, our second thermocycler, is mostly made out of a salvaged hairdryer
