Team:Duke
From 2009.igem.org
Sahilprasada (Talk | contribs) |
Sahilprasada (Talk | contribs) |
||
Line 186: | Line 186: | ||
<div id="home-box"> | <div id="home-box"> | ||
- | <p> The Duke University's iGEM team consists of 5 undergraduates, 3 graduates, and 2 professors. | + | <p> The Duke University's iGEM team consists of 5 undergraduates, 3 graduates, and 2 professors. Due to the high costs and inefficiency of the process of cloning a gene, the DUKE iGEM team has invented a new procedure which <b>lower costs</b> and also is <b>more efficient</b>. This method, Circular Polymerase Extension Cloning (CPEC), <b>saves time</b> as well, since this method does not involve ligation or restriction enzymes. The rising costs of the current method of producing biodegradable plastics has hindered its widespread use. However, this year's IGEM team has discovered a <b>more efficient pathway</b> to produce these biodegradable plastics. With this team's determination and motivation, they would like to present their two projects: <center> <b> CPEC applied to biobricks & Biodegradable Plastic Synthesis Pathway in E. coli. </center></b> |
+ | <br> <br> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
</p> | </p> |
Revision as of 02:20, 18 October 2009
Home | Project | Notebook | Team |
The Duke University's iGEM team consists of 5 undergraduates, 3 graduates, and 2 professors. Due to the high costs and inefficiency of the process of cloning a gene, the DUKE iGEM team has invented a new procedure which lower costs and also is more efficient. This method, Circular Polymerase Extension Cloning (CPEC), saves time as well, since this method does not involve ligation or restriction enzymes. The rising costs of the current method of producing biodegradable plastics has hindered its widespread use. However, this year's IGEM team has discovered a more efficient pathway to produce these biodegradable plastics. With this team's determination and motivation, they would like to present their two projects:
Project #1 - Biodegradable Plastic Synthesis Pathway in E. coli
Polyhydroxyalkanoic acids (PHA), naturally occurring storage polymers found in a variety of bacteria, have received increased attention for their potential use as bioplastics that are both biodegradable and reduce reliance on petroleum-based plastics. In particular, the copolymer poly(3-hyroxybutyrate-co-4-hydroxybutyrate), or poly(3HB-co-4HB), which combines the 3HB and 4HB polymers from different bacteria, has elastic properties ideal for a wide range of thermoplastic applications. The high cost of PHA, however, is the biggest impediment to widespread use of bioplastics. Moreover, poly(3HB-co-4HB) pathways developed so far in /E. coli/ have yielded undesirably low and unpredictable 4HB-to-3HB ratios.
Thus, this project aims to develop a more efficient biopathway for poly(3HB-co-4HB) while increasing the 4HB monomer composition predictably. It was hypothesized that optimizing codon permutations of the phaC gene would greatly increase affinity of PHA synthase to the 4HB monomer. To date, the phaCAB and cat2 operons have been cloned into pUC19 and PCR Blunt II-TOPO vectors for successful independent production of the 3HB and 4HB polymers. Ligation and transformation into /E. coli/ as six different recombinant constructs will soon be completed and allow for engineering of the poly(3HB-co4HB) biopathway. Future directions would be to test the hypothesis to see if phaC can be manipulated to increase 4HB-to-3HB composition in poly(3HB-co-4HB) and to increase efficient production of the bioplastic by engineering the FtsZ cell division protein to allow for cells to accumulate larger quantities of PHA granules before dividing. Ultimately, once an optimal biopathway is found, the goal would be to explore a model for mass production of PHA bioplastics so that novel applications of bioplastics can be feasible economically.
Project #2 - CPEC applied to biobricks
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.
|
Advisors
Dr. Jingdong Tian | jtian@duke.edu |
Faisal Reza | faisal.reza@duke.edu |
Students
Andrew Ang | aangandover@gmail.com |
Kevin Chien | kevin.chien@duke.edu |
Yaoyao Fu | yf21@duke.edu |
Faith Kung | fk8@duke.edu |
Sahil Prasada | sahil.prasada@duke.edu |
Jiayuan Quan | jq7@duke.edu |
Nicholas Tang | nicholas.tang@duke.edu |
Peter Zhu | peter.zhu@duke.edu |