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The process of high-throughput cloning is bottlenecked at the retriction and ligation stages. A combination of high costs, requirements for restriction site specific enzymes and general inefficiency of the process makes cloning on a large combinatorial gene library unviable. Circular Polymerase Extension Cloning (CPEC) addresses this issue by eliminating the need for restriction and ligation enzymes and thereby streamlining and condensing the procedure into the duration of 5 minutes. An extremely simple theory, CPEC piggybacks PCR in splicing genes. Figure 1 below outlines how CPEC may be used to insert a gene into a vector.

The gene insert is modified to have ends that overlap with the ends of the linearlized vector and both have similar melting temperatures. The insert and vector are placed within a PCR machine in the absence of primers. Denaturation separates the double-stranded insert and vector and the overlapping ends anneal. Polymerase extension mechanism is then used to complete the plasmid.

We will apply CPEC in the construction of a multi-component plasmid containing biobricks. Previous Duke iGEM projects have yielded the genes in a metabolic pathway that synthesizes poly(3HB-co-4HB), a biodegradable plastic, in E. coli. We will transform those genes into biobricks, with sticky ends, and efficiently combine them in a vector using CPEC.

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About our Team

Advisors

Dr. Jingdong Tian

Undergraduates

Sahil "Mr Baller" Prasada A remarkable individual of considerable talent, ability, and girth, the esteemed Mr. Prasada is the latest addition to Duke's elite iGEM team. His innumerable achievments span many varied fields, earning numerous recognitions at innovation summits (for his humor), at tennis tourneys (for his egregious sportsmanship), and at local dances (for his exaggerated gyrations of the hip) among others. His research interests are likewise broad, and as one of the best and brightest young minds of our time, he is very excited to share his latest findings with you at iGEM this November. Sahil aspires to be a cardiologist, just like his father, when he grows up. His favorite hobbies include tennis, kangaroo-watching, eating, and bhangra dancing. Sahil's academic interests include yeast mating, doxorubicin, and hydrogen fuels.

Kevin Chien A reformed Carolina fan.

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