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| − | <p>Click here to learn more about our design | + | <p>Click here to learn more about our project design</p> |
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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.
While present on a plasmid, the Hok-Sok system maintains plasmids through mRNA silencing. On the forward strand of the Hok-Sok cassette is the coding region for the Hok mRNA. Hok ("host killing") is a 52 amino acid toxin that is capable of permeating the cell membrane, causing a loss of electrochemical potential and cell death. The translation of Hok mRNA is constantly inhibited by Sok, an inhibitory RNA molecule that is transcribed on the reverse strand of the Hok-Sok cassette. Sok ("suppression of killing") is capable of forming a double-stranded RNA helix with the 5’-end of Hok mRNA, blocking the ribosome binding site and preventing translation. This RNA duplex is then recognized by RNAse III, leading to enzymatic degradation of the Hok transcript.
Below is a simplified diagram of how the Hok-Sok system maintains a plasmid:
How does this lead to plasmid maintenance? Hok mRNA, due to a high degree of secondary structure, has a long half-life, measured at 20 minutes. Sok, on the other hand, has a half-life of only 30 seconds. The cell must thus retain the coding region for Sok in order to produce enough Sok to continue blocking Hok translation. If the plasmid is lost, then both the Hok and Sok coding regions will be lost; however, previously transcribed Hok mRNA will still be present. With previously transcribed Sok rapidly degrading, Hok mRNA will be translated, killing the daughter cell that did not maintain the plasmid.
Our wet project for 2015 was to demonstrate that the Hok-Sok system could maintain a plasmid over many generations as well as typical antibiotic maintenance systems. We hypothesized that the Hok-Sok system could thus serve as a cheap, ethical alternative to antibiotic pressure.
PCR is a common tool used in the laboratory environment as it is a vital component in DNA amplification and any cloning procedure. However, the majority of PCR machines used in the lab are bulky and expensive and are rarely seen outside of biology-based labs. These issues inspired the development of Cheap Homemade Innovative PCR (or C.H.I.P. for short), a compact PCR machine that is as fast and proficient at amplifying DNA as lab based machines, but constructible for the fraction of the price of lab-grade PCR machines and a great tool for teaching-based applications. We are proud to say that we have been able to design a machine that will be completely open source and able to serve as a platform for DIY cloning procedures.
