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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.
Lutein
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.
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.
Lutein
Advanced Macular Degeneration is the current leading cause of blindness and vision loss in people aged 65 and over. 1.75 million people in the US are affected by AMD, and that number is expected to increase to almost 200 million worldwide by 2020. Lutein is a carotenoid currently used as a dietary supplement taken to treat and prevent the onset of AMD. Production of lutein is currently predominantly cultivation of marigolds. Carotenoids are extracted from its petals, from which lutein is then isolated.
Our aim is to introduce a system capable of producing lutein from carotenoid precursors into a bacterial system. As these pathways are native to plants and nonexistent in bacteria, the main challenge is obtaining every enzyme necessary to allow the pathway to occur. Strains of lycopene (a carotenoid precursor) producing bacteria already exist, and we expect to begin the synthesis from this point. Another main challenge is the regulation of genes required to proceed from lycopene to lutein. Lycopene is the precursor for a multitude of carotenoids, all of which are produced in plants due to necessity. To produce lutein, the lycopene must first undergo a reaction with the specific enzymes in order, ε-cyclase then β-cyclase. Having both enzymes in the system, though, allows reactions between β-cyclase and lycopene, which yields a product unable to be converted into lutein. Through regulatory measures such as altering gene expression levels, we plan to optimize the efficiency of the lutein synthesis pathway. We also aim to create a mathematical modeling system capable of correlating the expression levels of proteins to relevant efficiencies in similar synthesis pathways.
When people get old, some of them slowly lose their sight, like in the picture below. They will soon become blind. People can take a medicine called lutein to help treat their eyes and prevent the problem from getting worse. Right now, lutein is made from flowers. We want to find a cleaner and cheaper way to make lutein using 100% safe E. Coli. To do this, we will take DNA from plants that make lutein, and put that DNA into the E. Coli. Since there are many complicated chemicals involved, this task is not easy.
