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Revision as of 03:53, 8 September 2015
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.
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.
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.
PCR is often a common tool in the laboratory environment. It is also a vital component in DNA amplification and any cloning procedure. However the majority of theses devices are bulky expensive machines that are rarely seen outside of bio based labs. This was the premise for the development of CHIP or Cheap Homemade Innovative PCR, development of a compact PCR machine that was as fast and proficient at amplifying DNA as lab based machines but for the fraction of the price and 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.