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Team:Duke
From 2009.igem.org
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</div> | </div> | ||
| - | <div id="project-box"> <h1> Project </h1><p>The process of high-throughput | + | <div id="project-box"> |
| + | <h1> Project #1 - Biodegradable Plastic Biosynthesis Pathway in <i>E. coli</i> </h1> | ||
| + | <p>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. | ||
| + | <br><br> | ||
| + | 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.</p> | ||
| + | |||
| + | <h1> Project #2 - CPEC applied to biobricks</h1> | ||
| + | <p>The process of high-throughput | ||
cloning is bottlenecked at the retriction and ligation stages. A combination of | cloning is bottlenecked at the retriction and ligation stages. A combination of | ||
high costs, requirements for restriction site specific enzymes and general inefficiency | high costs, requirements for restriction site specific enzymes and general inefficiency | ||
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procedure into the duration of 5 minutes. An extremely simple theory, CPEC piggybacks | 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 | PCR in splicing genes. Figure 1 below outlines how CPEC may be used to insert | ||
| - | a gene into a vector.< | + | a gene into a vector. |
| + | <br><br> | ||
| + | The gene insert is modified to have ends that overlap | ||
with the ends of the linearlized vector and both have similar melting temperatures. | 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. | 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 | Denaturation separates the double-stranded insert and vector and the overlapping | ||
ends anneal. Polymerase extension mechanism is then used to complete the plasmid. | ends anneal. Polymerase extension mechanism is then used to complete the plasmid. | ||
| - | < | + | <br><br> |
| + | 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 | biobricks. Previous Duke iGEM projects have yielded the genes in a metabolic pathway | ||
that synthesizes poly(3HB-co-4HB), a biodegradable plastic, in <i>E. coli</i>. | that synthesizes poly(3HB-co-4HB), a biodegradable plastic, in <i>E. coli</i>. | ||
We will transform those genes into biobricks, with sticky ends, and efficiently | We will transform those genes into biobricks, with sticky ends, and efficiently | ||
| - | combine them in a vector using CPEC.</p></div> | + | combine them in a vector using CPEC.</p> |
| + | |||
| + | </div> | ||
| + | |||
| + | <!-- Notebook --> | ||
<div align="center" id="notebook-box"> | <div align="center" id="notebook-box"> | ||
Revision as of 21:00, 17 October 2009
| Home | Project | Notebook | Team |
Project #1 - Biodegradable Plastic Biosynthesis 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 |
