Team:Duke

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<div id="header"> <A HREF="http://www.bme.duke.edu/research/index.php">  
<div id="header"> <A HREF="http://www.bme.duke.edu/research/index.php">  
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<img src="http://www.duke.edu/~sp128/Header.jpg" alt="Duke logo" /> </A> </div>
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<img src="https://static.igem.org/mediawiki/2009/6/64/Header_revised.jpg" alt="Duke iGEM wiki header" /> </A> </div>
<table class="links">  
<table class="links">  
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  <td align="center" ><a id="show-home">Home</a></td>
  <td align="center" ><a id="show-home">Home</a></td>
  <td align="center" ><a id="show-project">Project</a></td>
  <td align="center" ><a id="show-project">Project</a></td>
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  <td align="center" ><a id="show-notebook">Notebook</a></td>
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                    <li><a href="javascript:;">Calendar</a> </li>
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                    <li><a href="javascript:;">Methods</a> </li>
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                    <li><a href="javascript:;">Protocol</a> </li>
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                <a class="links" style="background-color:none;" id="show-notebook">Notebook</a></li>
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</a></td>
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  <td align="center" ><a id="show-team">Team</a></td>
  <td align="center" ><a id="show-team">Team</a></td>
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<td align="center" ><a>Human Practices</a></td>
 
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</center>
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<div id="home-box"> <p>
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<center><font size="+1"><font color="blue"><b> One-Step Construction of a Bioplastic Production
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Pathway in E. <i>coli</i></b></font></font></center><br>
 +
<br>
 +
<b>We, the determined and motivated <font size="+1"><font color="blue">Duke University iGEM 2009 team</font></font> of 6 undergraduate students and 3 graduate students, have developed a practical and powerful method to deliver on the promise of sustainable <font size="+1"><font color="green">green</font></font> synthetic biology!</b><br>
 +
<br>
 +
Due to the high costs and inefficiency of the gene and pathway cloning process, we invented a new procedure which <font size="+1"><font color="blue">lowers costs</font></font> and <font size="+1"><font color="blue">increases efficiency</font></font>. This method, Circular Polymerase Extension Cloning (CPEC), <font size="+1"><font color="blue">saves time</font></font> as well, since it does not involve ligation or restriction enzymes.  See publication of our method <a href="http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006441">here.</a></font> <br>
 +
<br>
 +
We have applied our method to the production of <font size="+1"><font color="blue">biologically derived plastics</font></font>. The high costs of the current method of producing biodegradable plastics in an environmentally sound manner has hindered its widespread use.  However, we have discovered a <font size="+1"><font color="blue">more efficient pathway</font></font> to produce these biodegradable plastics. </p>
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<br><br>
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<center> <object width="580" height="360"><param name="movie" value="http://www.youtube.com/v/TZ_P8SIsWCQ&hl=en&fs=1&border=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/TZ_P8SIsWCQ&hl=en&fs=1&border=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="580" height="360"></embed></object></center><br>
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<div id="home-box"> <p> The Duke University's iGEM
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<div style="border:1.5px dashed black">
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team consists of 5 undergraduates, 3 graduates, and 2 professors. Due to the high
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<h2>Why do we deserve <b>GOLD</b>?</h2>
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costs and inefficiency of the process of cloning a gene, the DUKE iGEM team has
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<ul>
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invented a new procedure which <b>lowers costs</b> and also <b>increases efficiency</b>.
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<li> We had a <b>Great</b> summer working on our project</li>
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This method, Circular Polymerase Extension Cloning (CPEC), <b>saves time</b> as
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<li> We submitted <b>2</b> biobricks: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K282001">BBa_K282001</a> and <a href="http://partsregistry.org/Part:BBa_K282000">BBa_K282000</a></li>
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well, since this method does not involve ligation or restriction enzymes. The
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<li> We Created and implemented with great success a more accurate, flexible and efficient method of splicing multi-component biobricks systems into plasmid vectors. </li>
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rising costs of the current method of producing biodegradable plastics has hindered
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<li> and... </li>
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its widespread use; however, this year's IGEM team has discovered a <b>more efficient
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</ul>
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pathway</b> to produce these biodegradable plastics. With this team's determination
+
<br>
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and motivation, they would like to present their two projects: <center> <b> CPEC
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<center> <img src="https://static.igem.org/mediawiki/2009/a/a3/V_Duke.JPG" width="150" height="150"/> *We were also proud to help Team Valencia with their survey.</center>
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applied to biobricks & Biodegradable Plastic Synthesis Pathway in <i>E. coli</i>.
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</div>
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</B></center><br> <br> </p></div>
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 +
</div>
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<div id="project-box"> <h1> What is CPEC?</h1><p>
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<!-- Project -->
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Circular Polymerase Extension Cloning (CPEC) is the development of a much simplified  
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<div id="project-box">  
 +
<h1>Abstract</h1>
 +
A convenient ligation-free, sequence-independent one-step plasmid assembly and cloning method is developed [Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441]. This strategy, Circular Polymerase Assembly Cloning (CPEC), relies solely on polymerase extension to assemble and clone multiple fragments into any vector. Using this method, we are able to quickly assemble a metabolic pathway consisting of multiple enzymes and regulatory elements for the production of a biocompatible as well as biodegradable plastic polymer in E. <i>coli</i>.
 +
 
 +
<html>
 +
<h1> What is CPEC?</h1>
 +
 
 +
Circular Polymerase Extension Cloning (CPEC) is a much simplified  
sequence-independent cloning technology based entirely on the polymerase extension  
sequence-independent cloning technology based entirely on the polymerase extension  
mechanism. This method extends overlapping regions between the insert and vector  
mechanism. This method extends overlapping regions between the insert and vector  
-
fragments to form a complete circular plasmid. An extremely simple theory, CPEC
+
fragments to form a complete circular plasmid (Figure 1a). The basic principle of this strategy is that, after denaturation, both single-stranded vector and inserts overlap with each other by end sequences and extend by using the other as the primer in a typical PCR reaction. After complete circular plasmids with one nick in each strand are formed, the relaxed plasmids are transformed to competent cells and sealed as closed plasmids. CPEC strategy is not only suitable for the cloning of an individual gene but also for gene libraries, combinatorial libraries and multi-fragment plasmid assembling. With its simplicity, cost-effectiveness and high transformation efficiency, CPEC has become the most convenient, economical and accurate cloning method at the present day, especially for cloning applications with high complexity.  
-
piggybacks PCR in splicing genes. The gene insert is modified to have ends that  
+
-
overlap with the ends of the linearized 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.  
+
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<br>
+
-
 
+
</html>
</html>
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[[Image:CPEC.jpg|frame|center|500px|Figure 1. Circular Polymerase Extension Cloning]]
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 +
{| style="background:transparent; margin:auto"
 +
|-
 +
|| [[Image:CPEC.jpg|thumb|350px|Figure 1a. Biobrick spliced into vector using CPEC]]
 +
|| [[Image:multicomponent.jpg|thumb|350px|Figure 1b. Multicomponent Biobrick system spliced into vector simulatenously using CPEC]]
 +
|}
<html>
<html>
-
<H3> What are benefits?</H3>The process of high-throughput cloning is bottlenecked
 
-
at the restriction 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 inviable. 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.
 
<br>
<br>
-
<H3> Advantages/Disadvantages of BioBricks</H3><ul>
 
-
<li> BioBrick parts can be incorporated in <i> E. coli </i>, due to its common
 
-
interphase. </li><li> BioBrick parts are not easily made since its </li></ul><br>
 
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<h1>Standardized CPEC</h1>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 <i>E. coli</i>. We will transform those genes into biobricks, with sticky ends,
 
-
and efficiently combine them in a vector using CPEC.
 
-
<P></p>
 
-
<P></p>
 
-
<h3> Biodegradable  
+
<h1>One-Step Construction of a Bioplastic Production Pathway using CPEC</h1>
-
Plastic Synthesis Pathway in <i>E. coli</i></h3>
+
 
-
<p>Polyhydroxyalkanoic acids (PHA),
+
We applied CPEC in the construction of a multi-component
-
naturally occurring storage polymers found in a variety of bacteria, have received  
+
pathways in a single reaction. The proposed multi-way cloning mechanism is shown in Figure 1b. First, we constructed the biobrick parts containing genes involved in this pathway. Then we used multi-way CPEC for construction of
-
increased attention for their potential use as bioplastics that are both biodegradable
+
a metabolic pathway for synthesizing a biodegradable plastic, poly(3HB-co-4HB) in E. <i>coli.</i>
-
and reduce reliance on petroleum-based plastics. In particular, the copolymer
+
 
-
poly(3-hyroxybutyrate-co-4-hydroxybutyrate), or poly(3HB-co-4HB), which combines
+
<h3> Biodegradable Plastic Synthesis Pathway in E. <i>coli.</i></h3>
-
the 3HB and 4HB polymers from different bacteria, has elastic properties ideal
+
</html>[[Image:Fig.2a.jpg|framed|Figure 2. PHA granule production in bacteria]] <html>
-
for a wide range of thermoplastic applications. The high cost of PHA, however,
+
<p>Recently, polyhydroxyalkanoates (PHA) have received a lot of attention for their
-
is the biggest impediment to widespread use of bioplastics. Moreover, poly(3HB-co-4HB)  
+
potential use as bioplastic. They are naturally occurring biopolymers – under low nitrogen,
-
pathways developed so far in /E. coli/ have yielded undesirably low and unpredictable
+
phosphorus, or oxygen conditions, many bacteria produce protein- and lipid-coated granules of
-
4HB-to-3HB ratios.  
+
PHA for energy storage, and these granules can then in turn be harvested in large quantities as
 +
bioplastics (Fig. 2). Bioplastics not only help reduce the reliance on petroleum-based plastics, but
 +
also have a wide range of applications in the agricultural and medical fields because they are
 +
biodegradable and biocompatible. The most common PHA is poly(3-hydroxybutyrate), or poly(3HB), which is produced by
 +
the bacteria <i>Cupriavidus necator</i>. Three genes – phaA, phaB, and phaC – make up the
 +
phaCAB operon that synthesizes the enzymes required for formation of poly(3HB), which is not
 +
elastic enough for general plastic applications. But when phaCAB is expressed with cat2, a
 +
succinate degradation gene from Clostridium kluyveri that produces the 4HB monomer, the
 +
copolymer poly(3-hyroxybutyrate-co-4-hydroxybutyrate), or poly(3HB-co-4HB), is produced (Figure 3). Poly(3HB-co-4HB) is more elastic and thus has potential to be used in a variety of applications.  
 +
</html>
 +
 
 +
<html>
 +
However, bioplastic production is currently too costly to be used widely. For example,
 +
poly(3HB-co-4HB) pathways that have been developed so far produce copolymers with very low
 +
concentrations of 4HB, the monomer that gives the copolymer its elastic properties. This could
 +
be because the enzyme that catalyzes PHA formation, PHA synthase, has higher affinity for the
 +
3HB-CoA substrate (100%) than the 4HB-CoA substrate (0.03%). </html>
 +
[[Image:Bioplastic_pathway.jpg|framed|Figure 3. Pathway for poly(3HB-co-4HB) synthesis [Madison and Huisman, 1999]]]
 +
<html>In addition, the 3HB
 +
pathway uses acetyl-CoA, an input to the citric acid cycle, as a precursor, but cat2 uses succinyl-
 +
CoA, an intermediate in the cycle. Since more processing is required to obtain the 4HB cursor, the pathway may naturally favor production of the 3HB polymer, resulting in higher 3HB composition in the final copolymer.  
 +
 
<br><br>
<br><br>
-
Thus, this project aims to develop a more efficient  
+
Thus, this project aims to develop a more efficient biopathway for poly(3HB-co-4HB) while increasing the 4HB monomer composition predictably. To achieve this goal, we designed vectors with various combinations of the phaCAB operon and the cat2 gene so that genetic manipulations can be made to the genes for PHA synthase and precursor processing to
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biopathway for poly(3HB-co-4HB) while increasing the 4HB monomer composition predictably.  
+
increase bioplastic production and quality.
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It was hypothesized that optimizing codon permutations of the phaC gene would
+
</html>
-
greatly increase affinity of PHA synthase to the 4HB monomer. To date, the phaCAB
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[[Image:Fig.4.jpg|framed|Figure 4. The reddish tint present on some colonies indicates the presence of PHA granules in the bacteria]]
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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
+
<html>
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into /E. coli/ as six different recombinant constructs will soon be completed
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<h3>3HB genetic pathway</h3>
-
and allow for engineering of the poly(3HB-co4HB) biopathway. Future directions  
+
The phaCAB operon genomic DNA from <i>Cupriavidus necator</i> was cloned into PCR Blunt II-TOPO vector as the backbone and the cloning product was transformed into bacteria cells which were then plated on kanamycin (50 μg/ml) plates. Successful insertion of phaCAB was shown by Nile Red fluorescence in colonies (Figure 4).
 +
 
 +
<h3>4HB genetic pathway</h3>
 +
The cat2 gene was inserted into the pSOS vector to obtain the
 +
pSOS-cat2 construct. To obtain 4HB production, though, the phaC gene must also be expressed.
 +
The 4HB precursor &gamma;-butyrolactone (GBL) is required for 4HB production in this pathway, as the
 +
4HB precursor does not naturally exist in E. <i>coli.</i></html>
 +
 
 +
 
 +
 
 +
<html><h3>pASK vector constructs</h3>
 +
The PCR-Blunt II-phaCAB and pSOS-cat2 plasmids described above were then used as
 +
templates to create seven different vector constructs in pASK (Tables 1 and 2). The first
 +
six vector constructs were created by PCR amplification of the insert, restriction digestion of the
 +
vector and insert, ligation of the fragments, and finally transformation into E. <i>coli.</i> The seventh vector (pASKphaAB-term-pLZCat2phaC) was created using CPEC cloning and transformation into E. <i>coli</i> (Figure 5). All the constructs were sequenced to ensure that the constructs were correct. Aslo genotyping was done for the final construct 7 (Figure 6). </html>
 +
 
 +
{| class="wikitable" border="0" align="center"
 +
|-
 +
|[[Image:Bioplasticpathway_plasmid.jpg|thumb|380px|Figure 5. Construct 7.]]
 +
|[[Image:Multi_component_gel.jpg|thumb|380px|right|Figure 6. (Upper left) Gel electrophoresis analysis of the final
 +
assembly product after a 20-cycle CPEC. 5 ml of the reaction was
 +
separated on a 0.8% agarose gel and visualized after ethidium bromide
 +
staining. The full-length plasmid was 8360 bp. (Upper right) Gel electrophoresis
 +
analysis of the multi-way CPEC reaction. 5 ml was taken out of the
 +
reaction after 2, 5, and 10 cycles and separated on a 0.8% agarose gel
 +
(lanes 1, 2 and 3). The starting lengths of the four fragments were 3280,
 +
2959, 2040, and 171 bp, respectively. The 171-bp band was not visible.
 +
(Bottom) Restriction mapping of the isolated plasmids derived from the CPEC
 +
reaction. Plasmid DNA from five independent colonies (I-V) were
 +
digested with BamHI (lane 2, 8.4 kb), BamHI-XhoI (lane 3, 6.6 kb and
 +
1.8 kb), and NdeI (lane 4, 5.4 kb and 3 kb). Purified plasmids not
 +
subjected to restriction digestion are shown in lane 1. The molecular
 +
weight marker used in this figure was NEB 1 kb DNA ladder.]]
 +
|}
 +
 
 +
<html>
 +
<h3>Screening Experiments</h3>
 +
Two different experiments were conducted to investigate (1) the optimal
 +
anhydrotetracylcine level for inducing expression of bioplastic genes and (2) Nile Red as a
 +
screening technique for polymer production. To determine the optimal anydrotetracycline levels
 +
for phaCAB and cat2 gene expression, agar plates were prepared with 0, 5, 50, 150, and 200
 +
ng/ml of anyhdrotetracyline, and Construct 7 E. coli were grown on them overnight at 37°C. The
 +
number of colonies on each plate was then compared the following day. To test Nile Red as a
 +
screening technique to detect PHA production, agar plates were prepared with various levels of
 +
Nile Red (0, 0.2, 0.5,1.0 μg/ml). Bacteria that were known to produce PHA (positive control) and
 +
negative control bacteria were then plated and grown overnight at 37°C. After sitting on the
 +
benchtop for a few days, they were examined under equal levels of UV lighting.
 +
</html>
 +
[[Image:Fig.5.jpg|framed|Figure.7. Precipitated product from pASK-phaC-no tag (2 tubes on left) and negative control
 +
pASK (2 tubes on right) harvesting.]]
 +
 
 +
<html>
 +
 
 +
 
 +
<h3>Polymer harvesting</h3>
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To obtain in vivo PHA polymer, E. coli containing the desired construct were grown in 3
 +
mL of LB broth overnight, of which 1 mL was then cultured in 50 mL of LB broth and grown at
 +
30oC until the cells reached an optical density of .7 A (600 nm). Glucose was added to a final
 +
concentration of 1%, and the cells were harvested after 48 hour of incubation at 37°C.
 +
To harvest, the cells were centrifuged at 3500 rpm and suspended in saline (0.0067 M
 +
phosphate buffer) solution. The cells were re-centrifuged to obtain a washed pellet, freezethawed
 +
in dry ice, and then freeze-dried by lyophilization. The pellet was shaken overnight at 37°C in chloroform, after which bacterial cell waste was filtered out using Whatman paper and a vacuum flask. The polymer was then precipitated from the filtered solution by adding 10 volumes of ethanol, centrifuged, and then re-dissolved in chloroform. Finally, 10 volumes of methanol was used to re-precipitate the polymer before centrifuging to obtain a purified polymer pellet. A promising sign was that there was
 +
much more white powder precipitate in the experimental samples than the negative control
 +
samples after harvesting (Figure 7).
 +
 
 +
<h3><sup>1</sup>H NMR Spectroscopy</h3>
 +
To characterize harvested polymers, 300 MHz 1H NMR spectroscopy was used. The
 +
NMR sample was prepared by re-dissolving 20 mg of each polymer sample in 600 μl of
 +
deuterochloroform in an NMR tube. Attempts were made to harvest and identify the 3HB, 4HB, and poly(3HB-co-4HB)
 +
polymers. The first harvest attempt was made on pUC19phaCAB and then on pASK-phaCAB-no
 +
tag (Construct 1) to try and obtain 3HB polymer; the NMR results for the harvested product were
 +
compared to that of a negative control (no insert) and commercial 3HB. Several harvest attempts
 +
were also made on pSOS-cat2phaC bacteria (Construct 4) to try to confirm 4HB polymer
 +
production. Finally, bacteria transformed with pASKphaAB-termpLZCat2phaC (Construct 7)
 +
were harvested in an attempt to identify poly(3HB-co-4HB) by NMR.  
 +
</html>
 +
 
 +
 
 +
 
 +
{| class="wikitable" border="0" align="center"
 +
|-
 +
|[[Image:Fig.6.jpg|thumb|240px|Figure 8a. 300 MHZ <sup>1</sup>H NMR for commercial 3HB]]
 +
|[[Image:Fig.7.jpg|thumb|240px|Figure 8b. 300 MHZ <sup>1</sup>H NMR for experimental 3HB]]
 +
|[[Image:Fig.8.jpg|thumb|240px|right|Figure 8c. 300 MHZ <sup>1</sup>H NMR for negative control (pASK vector)]]
 +
|}
 +
 
 +
{| class="wikitable" border="0" align="center"
 +
|-
 +
|[[Image:Fig.9.jpg|thumb|380px|Figure 8d. Comparison of expansions of the methane proton region in the <sup>1</sup>H NMR spectra –commercial 3HB (left) and experimental (right).]]
 +
|[[Image:Fig.10.jpg|thumb|420px|right|Figure 8e. Comparison of expansions of the CH2 regions in the <sup>1</sup>H NMR spectra – Commercial 3HB (left) and experimental (right)]]
 +
|}
 +
 
 +
<html>The <sup>1</sup>H NMR spectra confirmed production of 3HB in the experimental bacteria and
 +
no 3HB production in the negative control bacteria (Fig. 8a-e). Commercial 3HB (from Sigma-
 +
Aldrich) was used as a comparison control (Fig. 8a). The 3HB peaks in the experimental sample
 +
matched the commercial 3HB sample, as well as 3HB spectra found in the literatures.
 +
However, the NMR spectra for the experimental and control samples had extremely large peaks
 +
from residual methanol and ethanol left over from the harvesting procedures, a problem
 +
corrected in later NMR readings by freeze-drying at the end of the harvesting procedure.
 +
 
 +
 
 +
Future directions  
would be to test the hypothesis to see if phaC can be manipulated to increase  
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  
4HB-to-3HB composition in poly(3HB-co-4HB) and to increase efficient production  
Line 311: Line 399:
to accumulate larger quantities of PHA granules before dividing. Ultimately, once  
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  
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  
+
production of PHA bioplastics so that novel applications of bioplastics can be </html>[[Image:Table 3.jpg|framed|Table 1. Vector designs with various phaCAB and cat2 combinations]]
 +
[[Image:Table 2.jpg|framed|Table 2. Restriction enzymes and details of the vector constructs]]
 +
<html>
feasible economically.</p>
feasible economically.</p>
-
</html>[[Image:Transformation.jpg|frame|center|500px|Figure 2]]<html>
+
</html>
-
</div>
 
-
<div align="center" id="notebook-box">
 
-
</html>
 
-
{{Imperial/Box1|Duke University iGEM Calendar|Below are several important milestones:
 
-
* [[Duke_University/30 October 2009|30]]-[[Duke_University/2 November 2009|2 November]]: iGEM Jamboree!|
 
-
<html><table style="background-color:#FFFFFF;"><tr><td></html>{{#calendar: title=Duke_University |year=2009 | month=06}}<html></td><td></html>{{#calendar: title=Duke_University |year=2009 | month=07}}<html></td><td></html>{{#calendar: title=Duke_University |year=2009 | month=08}}
 
-
<html></td></tr><tr><td></html>{{#calendar: title=Duke_University |year=2009 | month=09}}<html></td><td></html>{{#calendar: title=Duke_University |year=2009 | month=10}}<html></td><td></html>{{#calendar: title=Duke_University |year=2009 | month=11}}<html></td></tr></table><br><br></html>|}}
 
<html>
<html>
 +
<h1> Biobricks </h1>
-
<html>
+
In order to facilitate the completion of this project, several biobricks were created and used. The two foremost (the ones submitted) were phaAB and a double terminator.
-
<TABLE WIDTH="75%" BORDER="0"><TR> <TD BACKGROUND="https://static.igem.org/mediawiki/2009/0/0a/Paper.jpg"><BLOCKQUOTE><BLOCKQUOTE><BLOCKQUOTE><P ALIGN="LEFT">6/3/09
+
-
<BR>Experiment: PCR with contents of tube labeled 7 as DNA template, Gel with
+
-
100 bp ladder to check PCR <BR>Results: Incorrect ladder and template <BR>Conclusion:
+
-
Should use 1 kb ladder, Need to find correct template<BR> <BR>6/4/09<BR>E: Gel
+
-
to check Bioplastics #1-5<BR>R: All correct sized bands<BR>C: Use one as template
+
-
for PCR<BR> <BR>E: PCR with Bioplastics #5 as template<BR> <BR>5x HF buffer 5
+
-
ul <BR>dNTPs 2 ul<BR>forward primer 1.25 ul<BR>reverse primer 1.25 ul<BR>DNA template
+
-
(plasmid) 0.5 ul<BR>Phusion DNA polymerase 0.3 ul<BR>H2O 14.7 ul <BR> 25 ul<BR>
+
-
<BR>98&deg;C 30s 1x<BR>98&deg;C 15s <BR>55&deg;C 30s<BR>72&deg;C 1m 10s 35x<BR>72&deg;C
+
-
5m<BR>4&deg;C infinity 1x<BR> <BR>Fragment # Name Size/Length<BR>1 pASKphaA 4.2
+
-
kb <BR>2 phaB 900 bp <BR>3 Termcat2 1.7 kb<BR>4 phaC 1.9 kb<BR> <BR>6/5/09<BR>E:
+
-
Gel to check PCR<BR>R: Correct band for fragments 2 and 4, No band for fragments
+
-
1 and 3<BR>C: Cut correct bands out of gel, Try PCR with another template, different
+
-
conditions<BR> <BR>E: PCR with Bioplastics #1 as template, increased extension
+
-
time to 1m 20s<BR> <BR>6/6/09<BR>R: Correct band for fragments 1 (weak) and 2,
+
-
No band for fragments 3 and 4<BR>C: Cut out correct bands, Do PCR again with same
+
-
template for fragments 1 and 2, Try PCR with different template for fragments
+
-
3 and 4<BR> <BR>E: PCR with Bioplastics #1 (fragments 1 and 2) and #3 (fragments
+
-
3 and 4) as templates<BR> <BR> <BR>6/8/09<BR>E: Gel to check PCR, Gel with leftover
+
-
PCR of right sized fragments<BR>R: Correct bands for all fragments<BR>C: Cut out
+
-
bands<BR> <BR>6/9/09<BR>Gel Extraction<BR>1. Weigh cut gel piece(s). Add at least
+
-
1 ul binding buffer per mg of gel.<BR>2. Place tube at 50-60&deg;C for 10-15 min
+
-
or until gel is completely dissolved. Vortex every few minutes.<BR>3. Add solution
+
-
to spin column. Centrifuge at max speed for 30s. Discard filtrate.<BR>4. Add 300
+
-
ul binding buffer to column. Centrifuge for 30s. Discard filtrate.<BR>5. Add 500
+
-
ul wash buffer. Centrifuge for 30s. Discard filtrate.<BR>6. Repeat step 5.<BR>7.
+
-
Centrifuge tubes for at least 2 min. Discard collection tube.<BR>8. Obtain 1.7
+
-
ml centrifuge tube. Add 40-50 ul elution buffer to spin column. Centrifuge for
+
-
1 min. Discard spin column.<BR> <BR>E: Measured concentration of DNA fragments<BR>R:
+
-
Very low concentrations of fragments 1 and 3<BR>C: Do PCR with fragments as template,
+
-
greater volume to increase amount<BR> <BR>E: PCR with fragments 1 and 3 as templates</P><P ALIGN="LEFT">6/10/09
+
-
<BR>E: Gel to check PCR <BR>R: Smears at bottom of gel <BR>C: Should use Bioplastics
+
-
#1 as template to increase amount of fragment 1 and Bioplastics #3 as template
+
-
to increase amount of fragment <BR> <BR>E: PCR with Bioplastics #1 and #3 as templates
+
-
and doubled volume, Gel to obtain more of fragments<BR>R: Smears for fragment
+
-
1, Correct bands for fragment 2, Correct bands and smears for fragment 3 <BR>C:
+
-
PCR with different conditions <BR> <BR>E: PCR with Bioplastics #3 as template,
+
-
extension time increased to 1m 30s, cycles increased to 40 <BR> <BR>6/11/09 <BR>E:
+
-
Gels to check PCR <BR>R: Correct bands for fragment 1, 2 and 4 and possibly correct
+
-
band for fragment 3 <BR>C: Cut out bands<BR> <BR>E: Measured concentration of
+
-
DNA fragments<BR>R: Still low concentrations of fragments 1 and 3<BR>C: Do more
+
-
PCRs to obtain more of fragments 1 and 3</P><P ALIGN="LEFT"><BR>6/15/09 <BR>E:
+
-
PCRs with Bioplastics #1 (fragments 1 and 2) and #3 (fragments 3 and 4) as templates,
+
-
doubled volume, eliminated annealing step, varied extension (plus annealing) time
+
-
based on fragment size, Gels to check PCRs<BR> <BR>5x HF buffer 10 ul <BR>dNTPs
+
-
4 ul<BR>forward primer 2.5 ul<BR>reverse primer 2.5 ul<BR>DNA template (plasmid)
+
-
1 ul<BR>Phusion DNA polymerase 0.8 ul<BR>H2O 29.2 ul <BR> 50 ul<BR> <BR>98&deg;C
+
-
30s 1x<BR>98&deg;C 15s <BR>72&deg;C varies 35x<BR>72&deg;C 5m<BR>4&deg;C infinity
+
-
1x<BR> <BR>Annealing Time: 30s <BR>Extension Time: 15 s * size/length (kb)<BR>
+
-
<BR>Fragment # Name Size/Length (bp) Time (s)<BR>1 pASKphaA 4215 100<BR>2 phaB
+
-
896 50<BR>3 Termcat2 1701 60<BR>4 phaC 1872 65<BR> <BR>R: Bands for fragments
+
-
1 and 2, No bands for fragments 3 and 4<BR>C: Cut out bands of fragments 1 and
+
-
2, PCR again to obtain fragments 3 and 4<BR> <BR>6/16/09<BR>E: PCR with Bioplastics
+
-
#2 as template and Phusion mix (done by Maggie), Gels to check PCRs<BR> <BR>2x
+
-
Phusion mix 25 ul<BR>forward primer 2.5 ul<BR>reverse primer 2.5 ul<BR>DNA template
+
-
(plasmid) 1 ul<BR>H2O 19 ul<BR> 50 ul<BR> <BR>R: No bands for fragments 3, Bands
+
-
for fragment 4<BR>C: Cut out bands of fragment 4, PCR again to obtain fragment
+
-
3<BR> <BR>E: Measured concentration of DNA fragments 1 and 2<BR>R: Concentrations
+
-
okay<BR>C: Do assembly PCR once enough of fragments 3 and 4 obtained<BR> <BR>6/17/09<BR>E:
+
-
PCR with Bioplastics #4 as template, using Phusion enzyme protocol (shorter denaturation
+
-
time, lower annealing temperature), Gel to check PCRs<BR> <BR>98&deg;C 15s 1x<BR>98&deg;C
+
-
1s<BR>50&deg;C 5s <BR>72&deg;C 30s 35x<BR>72&deg;C 2m<BR>4&deg;C infinity 1x<BR>
+
-
<BR>R: Correct bands<BR>C: Cut out bands<BR> <BR>E: Measured concentration of
+
-
DNA fragments 3 and 4<BR>R: Concentrations high<BR>C: Do assembly PCR</P><P ALIGN="LEFT"><BR>6/18/09
+
-
<BR>E: Assembly PCR, Gels to check PCRs <BR> <BR>2x Phusion mix 12.5 ul <BR>insert
+
-
7.14 ul <BR>H2O 5.36 ul <BR> 25 ul <BR> <BR>98&deg;C 30s 1x<BR>98&deg;C 10s<BR>70-55&deg;C
+
-
5s <BR>72&deg;C 30s 25x<BR>72&deg;C 2m<BR>4&deg;C infinity 1x</P><P ALIGN="LEFT">Fragment
+
-
# Name Size/Length (bp) Concentration (ng/ul) Mass (ng) Volume (ul)<BR>1 pASKphaA
+
-
4215 41 194 4.73<BR>2 phaB 896 68 41 0.60<BR>3 Termcat2 1701 69 78 1.13<BR>4 phaC
+
-
1872 126 86 0.68<BR> ~400 7.14<BR> <BR>Length of plasmid: 8360 bp + 4 Histags
+
-
* 6 aa * 3 bp = 8432 bp</P><P ALIGN="LEFT">R: Smear because of misformed gel,
+
-
no band<BR>C: Change conditions<BR> <BR>6/19/09<BR>E: Assembly PCR (done by Maggie)
+
-
with different conditions (decreased extension time, increased number of cycles)<BR>
+
-
<BR>5x HF buffer 5 ul <BR>dNTPs 2 ul<BR>insert 7.18 ul<BR>Phusion DNA polymerase
+
-
0.4 ul<BR>H2O 10.42 ul <BR> 25 ul<BR> <BR>98&deg;C 30s 1x<BR>98&deg;C 10s<BR>70-55&deg;C
+
-
30s <BR>72&deg;C 2m 40x<BR>72&deg;C 5m<BR>4&deg;C infinity 1x<BR> <BR>Fragment
+
-
# Name Mass (ng) Volume (ul)<BR>1 pASKphaA 195.3 4.76<BR>2 phaB 41 0.60<BR>3 Termcat2
+
-
78.4 1.14<BR>4 phaC 86.4 0.68<BR> ~400 7.14<BR>6/20/09 <BR>E: Gel to check PCR<BR>R:
+
-
Correct band (faint)<BR>C: Low efficiency, Use different method (Infusion kit
+
-
or PCR with 2 fragments first)<BR> <BR>6/22/09<BR>LB Agar Plates with Chlorophenicol
+
-
(CAM), anhydrotetracycline, Nile Red (NR)<BR> stock plate<BR>CAM 50 mg/ul 20 ug/ml<BR>anhyd
+
-
2 mg/ml 150 ng/ml<BR>NR 0.25 mg/ml 0.5 ug/ml<BR> <BR>E: PCR with fragments 1 and
+
-
2 and different conditions (decreased annealing and extension times and number
+
-
of cycles), Gel to check PCR<BR> <BR>2x Phusion mix 12.5 ul <BR>pASKphaA 4.76
+
-
ul<BR>phaB 0.60 ul<BR>H2O 7.14 ul <BR> 25 ul<BR> <BR>98&deg;C 30s 1x<BR>98&deg;C
+
-
10s<BR>70-55&deg;C 10s <BR>72&deg;C 1m 10s 20x<BR>72&deg;C 5m<BR>4&deg;C infinity
+
-
1x<BR> <BR>R: No band/smear<BR>C: Change conditions<BR> <BR>6/23/09<BR>E: Assembly
+
-
PCR with different conditions (no slow ramp)<BR> <BR>6/24/09<BR>E: Gel to check
+
-
PCR<BR>R: Correct band (faint)<BR>C: Do transformation with competent cells</P><P ALIGN="LEFT"><BR>6/24/09
+
-
<BR>Transformation with High Efficiency GC5 Competent Cells <BR>1. Remove competent
+
-
cells from -70&deg;C and place on ice. Thaw for 5-10 min. <BR>2. Gently mix cells
+
-
by tapping tube. <BR>3. Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl
+
-
pipette tip while dispensing DNA. Gently tap tube to mix. <BR>4. Place tubes on
+
-
ice for 30 min. <BR>5. Heat-shock cells for 45 sec in 42&deg;C (water) bath. Do
+
-
not shake! <BR>6. Add 450 ul RT SOC Medium to each transformation reaction. <BR>7.
+
-
Incubate at 37&deg;C for 1 hr with shaking at 225-250 rpm. <BR>8. Spread on LB
+
-
agar plates containing appropriate antibiotic.<BR>9. Incubate plates at 37&deg;C
+
-
overnight (12-16 hrs). <BR> <BR>6/26/09<BR>Pick a colony from a streaked selective
+
-
plate to inoculate 10 ml of LB medium supplemented with the appropriate selection
+
-
antibiotic. Incubate 12-18 hrs at 37&deg;C while shaking at 200-250 rpm.<BR> <BR>6/27/09<BR>Harvest
+
-
bacterial culture by centrifuging at 8000 rpm (6800xg) in microcentrifuge for
+
-
2 min at RT. Decant supernatant and remove remaining medium.<BR> <BR>Purification/Mini
+
-
prep<BR>1. Resuspend pelleted cells in 250 ul Resuspension Solution. Vortex or
+
-
pipet up and down until no cell clumps remain.<BR>2. Add 250 Lysis Solution and
+
-
mix thoroughly by inverting tube 4-6 times until solution becomes viscous and
+
-
slightly clear. Do not incubate for more than 5 min to avoid denaturation of supercoiled
+
-
plasmid DNA.<BR>3. Add 350 ul Neutralization Solution and mix thoroughly by inverting
+
-
tube 4-6 times. <BR>4. Centrifuge for 5 min to pellet cell debris and chromosomal
+
-
DNA.<BR>5. Transfer supernatant to spin column by decanting or pipetting. Avoid
+
-
disturbing or transferring white precipitate.<BR>6. Centrifuge for 1 min. Discard
+
-
flow-through and replace column in collection tube.<BR>7. Add 500 ul Wash Solution
+
-
to spin column. Centrifuge for 30-60s and discard flow-through.<BR>8. Repeat step
+
-
7.<BR>9. Centrifuge for 1 min to remove residual Wash Solution.<BR>10. Transfer
+
-
spin column into fresh 1.7 ml microcentrifuge tube. Add 50 ul Elution Buffer to
+
-
center of spin column membrane. Do not touch pipette tip to membrane. Incubate
+
-
2 min at RT and centrifuge for 2 min.<BR>Note: An additional elution step with
+
-
Elution buffer or water will recover residual DNA from the membrane and increase
+
-
overall yield by 10-20%.<BR>11. Discard column and store purified plasmid DNA
+
-
at -20&deg;C. <BR> <BR>6/29/09 <BR>E: Restriction digests, Gel to check digests<BR>
+
-
<BR>Digest 1<BR>NdeI (FastDigest) 1 ul<BR>SpeI (NEB) 1 ul<BR>10x FastDigest buffer
+
-
2 ul<BR>plasmid 1 ul (~100 ng)<BR>H2O 15 ul<BR> 20 ul<BR>Band Sizes/Lengths: 5
+
-
kb, 3 kb, 460 bp, (80bp)<BR> <BR>Digest 2 (Did not do)<BR>NdeI (FastDigest) 2
+
-
ul<BR>10x FastDigest buffer 2 ul<BR>plasmid 1 ul (~100 ng)<BR>H2O 15 ul<BR> 20
+
-
ul<BR>Band Sizes/Lengths: 5.4 kb, 3 kb<BR> <BR>Digest 3<BR>BamHI (FastDigest)
+
-
1 ul<BR>XhoI (FastDigest) 1 ul<BR>10x FastDigest buffer 2 ul<BR>plasmid 1 ul (~100
+
-
ng)<BR>H2O 15 ul<BR> 20 ul<BR>Band Sizes/Lengths: 6.6 kb, 1.8 kb<BR> <BR>Digest
+
-
4<BR>BamHI (FastDigest) 2 ul<BR>10x FastDigest buffer 2 ul<BR>plasmid 1 ul (~100
+
-
ng)<BR>H2O 15 ul<BR> 20 ul<BR>Band Size/Length: 8.4 kb<BR> <BR>R: Correct bands
+
-
and sizes for plasmid 2 and digests 1, 3, and 4, correct band and size for plasmid
+
-
4<BR>C: Do more tests on plasmid 2 such as sequencing to determine if plasmid
+
-
is carryover, Try different primers</P></BLOCKQUOTE></BLOCKQUOTE></BLOCKQUOTE></TD></TR></TABLE><P ALIGN="LEFT">&nbsp;</P><P ALIGN="LEFT"><BR></P><br>
+
 +
<br>
 +
<h3> phaAB </h3>
 +
<br>
 +
Part: <b><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K282000"> BBa_K282000 </a></b>
 +
<br>
 +
Used in the creation of bioplastics. Contains both the PhaA gene and the PhaB gene. Part of the pathway PhaAB - Terminator - Cat2PhaC .
 +
<br>
 +
 +
<br>
 +
<h3> Terminator </h3>
 +
<br>
 +
Part: <b><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K282001"> BBa_K282001 </a></b>
 +
<br>
 +
 +
Based off of Part:<b>BBa_B0014</b>.  Comprised of forward terminator Part:<b>BBa_B0010</b> and reverse terminator Part:<b>BBa_B0012</b>.
 +
 +
<br>
</div>
</div>
-
<div id="team-box">
+
<!-- Notebook -->
-
<h2>Advisors</h2>
+
 
-
<TABLE WIDTH="75%" BORDER="0"> <TR>  
+
<div id="notebook-box"></html>
-
<TD WIDTH="37%">Dr. Jingdong Tian</TD><TD WIDTH="63%">jtian@duke.edu</TD></TR>  
+
 
-
<TR> <TD WIDTH="37%">Faisal Reza </TD><TD WIDTH="63%">faisal.reza@duke.edu</TD></TR></TABLE><br />  
+
=Calendar=
-
<h2>Students<BR></h2>
+
<html><center><iframe src="http://www.google.com/calendar/embed?showPrint=0&amp;mode=AGENDA&amp;height=600&amp;wkst=1&amp;bgcolor=%23FFFFFF&amp;src=aoqcbepvqdo8afcgih0d5mj1cc%40group.calendar.google.com&amp;color=%23A32929&amp;ctz=America%2FNew_York" style=" border-width:0 " width="900" height="600" frameborder="0" scrolling="no"></iframe></center></html>
-
</html>
+
 
 +
=Protocols=
 +
 
 +
==CPEC Cloning==
 +
[[#Protocols | Back to top]]
 +
 
 +
'''Materials'''
 +
* Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
 +
* Thermocycler
 +
 
 +
'''Preparation'''
 +
<pre>
 +
5x Phusion HF Buffer    4 ul
 +
10 mM dNTPs         0.4 ul
 +
Vector         50 ng/1kb
 +
Insert         x ng*
 +
Phusion DNA Polymerase  0.2 ul
 +
-------------------------------
 +
H2O         to 20 ul
 +
</pre>
 +
 
 +
<nowiki>*The amount of insert is determined so that the molar ratio for vector and insert is 1 to 2.</nowiki>
 +
 
 +
'''Procedures'''
 +
<pre>
 +
98°C                  30sec
 +
  10X
 +
    98°C            10 sec
 +
    Annealing**      30 sec
 +
    72°C            x sec***
 +
72°C                  5min
 +
4°C                  hold
 +
</pre>
 +
 
 +
<nowiki>** Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.</nowiki><br />
 +
<nowiki>***The extension time is usually calculated according to the shortest piece with 15 sec /kb if the cloning is not complicated. For example, if there is only one insert and is shorter than the vector, say, 600 bp, then I will use 15 sec for extension. Refer to the published paper for detailed information.</nowiki>
 +
 
 +
==DNA Purification==
 +
[[#Protocols | Back to top]]<br />
 +
 
 +
'''Materials :''' <html> <ul>
 +
<li> E.Z.N.A Gel Purification Kit (Omega Bio-Tek, Cat No. D2500-02 )</li>
 +
<li> Water bath equilibrated to 55-65C</li>
 +
<li>Microcentrifuge capable of at least 10,000 x g</li>
 +
<li>Nuclease-free 1.5 ml centrifuge bottles</li>
 +
<li>Absolute (95%-100%) ethanol</li>
 +
<li>Protective eye-wear</li>
 +
<li>Isopropanol (for fragments < 500 bp only)</li></ul></html>
 +
 
 +
'''Protocol:'''
 +
 
 +
# Perform agarose gel electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended, however, that fresh TAE buffer or TBE buffer be used as running buffer. Do not re-use running buffer as its pH will increase and reduce yields.
 +
# When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean scalpel.
 +
# Determine the approximate volume of the gel slice by weighing it in a clean 1.5 ml microfuge tube. Assuming a density of 1 g/ml of gel, the volume of gel is derived as follows: A gel slice of mass 0.3 g will have a volume of 0.3 ml. Add equal volume of Binding Buffer (XP2). Incubate the mixture at 55C-60C for 7 min or until the gel has completely melted. Mix by shaking or inverting the tube every 2-3 minutes. Centrifuge the tube briefly to collect all the liquid to the bottom of the tube.
 +
 
 +
Note: For DNA fragment less than 500bp, add 1 sample volume of isopropanol after the addition of Binding Buffer (XP2).
 +
 
 +
# Apply up to 700 ul of the DNA/agarose solution to a HiBind® DNA spin column assembled in a clean 2 ml collection tube (provided) and centrifuge in a microcentrifuge at 8,000-10,000 x g for 1 min at room temperature. Discard the liquid. Re-use the collection tube in Steps 5-8. For volumes greater than 700 ul, load the column and centrifuge successively, 700 ul at a time. Each HiBind® spin-column has a total capacity of 25-30 ug DNA.
 +
# Discard liquid and add 300ul Binding Buffer. Centrifuge at 10,000 x g for 1 minutes.
 +
# Add 700 ul of SPW Buffer diluted with absolute ethanol into the column and wait 2-3 minutes. Centrifuge at 10,000 x g for 1 min at room temperature to wash the sample.
 +
# Discard liquid and repeat step 6 with another 700 ul SPW Buffer.
 +
# Discard liquid and, re-using the collection tube, centrifuge the empty column for 1 min at maxi speed (>13,000 x g)  to dry the column matrix. This drying step is critical for good DNA yields.
 +
# Place column into a clean 1.5 ml microcentrifuge tube (not provided). Add 30-50 ul depending on desired concentration of final product) Elution Buffer (or sterile deionized water) directly to the center of the column matrix, then incubate for 1 minute. Centrifuge 1 min at maxi speed (>13,000 x g) to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lo
 +
 
 +
==PCA (Polymerase Cycle Assembly)==
 +
[[#Protocols | Back to top]] <br />
 +
 
 +
'''Materials'''
 +
* Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
 +
* Thermocycler
 +
 
 +
'''Preparation'''
{|
{|
-
|[[Image:Anga.jpg|250px]]
+
|5x Phusion HF Buffer || 5 ul
-
|'''Andrew Ang''' <br /> andrew.ang@duke.edu <br /> bio
+
|-
|-
-
|[[Image:chienk.jpg|250px]]
+
|10 mM dNTPs || 0.5 ul
-
|'''Kevin Chien''' <br /> kevin.chien@duke.edu <br />
+
-
Kevin S. Chien was a singular inspiration in the lives of all whom he knew. His ever-present cheer, his quiet genius, and his genuine concern for the well-being of others all served to make his existence an indelible influence upon the memories of others. Kevin’s contagious enthusiasm and love for life were apparent in all he did—whether it was music, lacrosse (of which he was an avid fan and participant), or Frisbee, Kevin’s whole-hearted commitment provided stellar, inspiring leadership. As a bright light in the lives of all those who he loved and loved him, he will be missed but remembered by all as an upright, selfless, warm friend. He is currently well, in attendance at Duke University, and will be attending iGEM 2009.
+
|-
|-
-
|[[Image:Faith.jpg|250px]]
+
|Oligo mixture || 125 ng /250 ng /500 ng /
-
|'''Faith Kung''' <br /> fk8@duke.edu <br />
+
|-
-
Kung is a senior at Duke majoring in Biomedical Engineering with minors in Music  
+
|Phusion DNA Polymerase || 0.25 ul
-
Performance and Biology. She enjoys working in a lab. Besides academics, her hobbies  
+
|-
-
include arts and crafts, dance, and figure skating. Faith is applying to PhD programs  
+
| --------------------- || --------
 +
|-
 +
|H2O || to 25 ul
 +
|}
 +
 
 +
 
 +
'''Procedures'''
 +
<pre>
 +
98°C              30sec
 +
  40X
 +
      98°C        7 sec
 +
      70-50°C    slow ramp, 0.1°C/sec
 +
      50°C        30 sec
 +
      72°C        15 sec /kb
 +
72°C              5 min
 +
4°C              hold
 +
</pre>
 +
 
 +
==PCR Product Clean-up for DNA Sequencing==
 +
[[#Protocols | Back to top]]
 +
 
 +
'''Materials:'''
 +
* ExoSAP-IT® (usb, Cat. No. 78200)
 +
*Thermocycler
 +
 
 +
'''Protocol:'''
 +
# Remove ExoSAP-IT from -20°C freezer and keep on ice throughout this procedure.
 +
# Mix 5 μl of a post-PCR reaction product with 2 μl of ExoSAP-IT for a combined 7 μl reaction volume.
 +
# Incubate at 37°C for 15 min to degrade remaining primers and nucleotides.
 +
# Incubate at 80°C for 15 min to inactivate ExoSAP-IT.
 +
# The PCR product is now ready for use in DNA sequencing etc.
 +
 
 +
==PCR==
 +
[[#Protocols | Back to top]]
 +
 
 +
'''Materials'''
 +
* Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
 +
* Thermocycler
 +
 
 +
'''Preperation'''
 +
{|
 +
|-
 +
|5x Phusion HF Buffer || 10 ul
 +
|-
 +
|10 mM dNTPs || 1 ul
 +
|-
 +
|DNA template || 1 pg – 10 ng
 +
|-
 +
|Forward primer (10 uM) || 2.5 ul
 +
|-
 +
|Reverse primer (10 uM) || 2.5 ul
 +
|-
 +
|Phusion DNA Polymerase || 0.5 ul
 +
|-
 +
| -------------------- || -----
 +
|-
 +
|H2O || to 50 ul
 +
|}
 +
 
 +
 
 +
 
 +
'''Procedure'''
 +
<pre>
 +
98°C          30sec
 +
30X
 +
    98°C      10 sec
 +
    Annealing* 30 sec
 +
    72°C      15 sec per 1 kb
 +
72°C          5min
 +
4°C            hold
 +
</pre>
 +
 
 +
 
 +
<nowiki>* Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.</nowiki>
 +
 
 +
==Single Colony PCR==
 +
[[#Protocols | Back to top]]
 +
 
 +
'''Materials'''
 +
* Taq DNA Polymerase with Standard Taq Buffer (NEB, Cat. No. M0273)
 +
* 10 mM dNTP Mix (NEB, Cat. No. N0447)
 +
* Thermocycler
 +
 
 +
'''Preperation'''
 +
{|
 +
|-
 +
|10x Standard Taq Buffer || 2 ul
 +
|-
 +
|10 mM dNTPs || 0.4 ul
 +
|-
 +
|Bacteria culture* || 1 ul
 +
|-
 +
|Forward primer (10 uM) || 1 ul
 +
|-
 +
|Reverse primer (10 uM) || 1 ul
 +
|-
 +
|Taq DNA Polymerase || 0.2 ul
 +
|-
 +
| ----------------- || -------
 +
|-
 +
|H2O || to 20 ul
 +
|}
 +
<nowiki>*Bacteria culture refers to E. coli cultured in LB solution overnight.</nowiki>
 +
 
 +
'''Procedures'''
 +
<pre>
 +
94°C              5 min
 +
25X
 +
    94°C        15 sec
 +
    Annealing**  30 sec
 +
    72°C        1 min per kb
 +
72°C              5min
 +
4°C              hold
 +
</pre>
 +
 
 +
<nowiki>**Anneal at Tm which is calculated with salt-adjusted method.</nowiki>
 +
 
 +
==Transformation==
 +
[[#Protocols | Back to top]] <br />
 +
 
 +
'''Materials:'''<html><ul>
 +
<li>GC5 Chemical Competent Cells (Genesee Scientific, Cat. No. 42-653)</li>
 +
<li>SOC Medium (Sigma, Cat. No. S1797)</li>
 +
<li>LB Agar (Sigma, Cat. No. L3027)</li>
 +
<li>Petri Dishes (VWR, Cat. No. SC25373-187)</li>
 +
<li>Cell Spreader (VWR, Cat. No. 89042-018)</li>
 +
<li>37°C incubator</li>
 +
<li>37°C shaker</li>
 +
<li>water bath</li></ul></html>
 +
 
 +
'''Protocol:'''
 +
 
 +
1. Thaw 1 tube of competent cells on ice;
 +
 
 +
2. Add 3 ul of cloning product or 1-50 ng of plasmid into competent cells while stirring gently;
 +
 
 +
3. Keep the tube covered by ice for 30min;
 +
 
 +
4. Heat-shock the competent cells in water bath for 45 sec at 42°C;
 +
 
 +
5. Put the tube on ice for 2 minutes;
 +
 
 +
6. Add 450 ul of SOC medium and then put it in a 37°C shaker for 1 hour;
 +
 
 +
7. Dilute and spread an appropriate amount on an LB agar plate with the appropriate antibiotics;
 +
 
 +
8. Place the plate up-side-down in 37°C incubator for 16-18 hours (overnight).
 +
 
 +
==NMR==
 +
 
 +
# Login and start NMR program.
 +
# Click Acqi. Load sample. Make sure spin is on and lock is off.
 +
# Go to lock. Decrease number of sine waves to 1 to obtain step function. Make sure Zo is 1100, lockpower is below 30, lockgain is 36, lockphase is 352, and spin is 20.
 +
# Go to shim. Increase lock level.
 +
# Click main menu. Click set up. Click H1CDCl3. Type ‘nt=64’ (number of scans), ‘ss=2’ (dummy scans), ‘go’, ‘lb=0.2’ (line broadening).
 +
# Viewing: Type ‘wft’, ‘dscale’, ‘dfp’ to display peak frequency.
 +
# Zooming in: Click display. Go to interactive. Type ‘cr=8p, delta=8p’ to set width of zoom. Use cursor to set boundaries. Click to cut.
 +
# Printing: Type “pL pscale(0) pltext ppf page’ to print spectra with text and peak frequencies.
 +
 
 +
= Lab Safety =
 +
 
 +
None of our project ideas raised any safety issues whether it be researcher, environmental, or public safety.  Duke has an in-house review board for projects and they did not raise any flags regarding our project.  The biobrick parts created also do not pose any safety issues.
 +
 
 +
 
 +
<html>
 +
</div>
 +
 
 +
 
 +
<!-- Team Profile -->
 +
 
 +
<div id="team-box"></html>
 +
 
 +
[[Image:IMG_2374.jpg|thumb|400px|Not Including: Andrew Ang and Kevin Chien]]
 +
 
 +
 
 +
<html><h2>Students</h2></html>
 +
{|
 +
|[[Image:Anga.jpg|200px]]
 +
|'''Andrew Ang''' <br /> andrew.ang(at)duke.edu <br /> Andrew Ang is a freshman at Duke, majoring in Biomedical Engineering. Apart from class and iGEM, he is involved in the Jazz Ensembles program and Asian Students Association at Duke. His hobbies include piano, saxophone, tennis, and squash. He is interested in molecular and synthetic biology, biomolecular engineering and medical research. He has previously worked as part of the MIT 2008 team, and he is excited to participate in iGEM again this year, and many more years to come.
 +
|-
 +
|[[Image:chienk.jpg|200px]]
 +
|'''Kevin Chien''' <br /> kevin.chien(at)duke.edu <br />
 +
Kevin Chien is a freshman at duke, majoring in Biomedical Engineering. He wants to explore his options in many different careers. He occasionally plays Club Frisbee and is interested in many different areas of knowledge, ranging from Astrophysics to Public Policy (and even sometimes History). He worked on this iGEM team last year as well and enjoyed his stay in Boston(hopes to have a good one this year too!).
 +
|-
 +
|[[Image:Fuy.jpg|200px]]
 +
|'''Yaoyao Fu''' <br /> yf21(at)duke.edu <br />
 +
Yaoyao Fu is a master's student in Biomedical Engineering. She is interested in molecular synthetic biology. Her hobbies are traveling and sports.
 +
|-
 +
|[[Image:Faith.jpg|200px]]
 +
|'''Faith Kung''' <br /> fk8(at)duke.edu <br />
 +
Faith Kung is a senior at Duke majoring in Biomedical Engineering with minors in Music and Biology. She enjoys working in a lab. Besides academics, her hobbies  
 +
include arts and crafts, dance, and figure skating. Also, she is actively involved in the IV Christian Fellowship. Faith is applying to PhD programs  
in Biomedical Science and hopes to pursue a career in scientific research and  
in Biomedical Science and hopes to pursue a career in scientific research and  
education. She is excited about attending the iGEM competition this year.
education. She is excited about attending the iGEM competition this year.
|-
|-
-
|[[Image:PrasadaS.jpg|250px]]
+
|[[Image:PrasadaS.jpg|200px]]
-
|'''Sahil "Most Valuable Baller" Prasada''' <br /> sahil.prasada@duke.edu <br />
+
|'''Sahil Prasada''' <br /> sahil.prasada(at)duke.edu <br />
Sahil Prasada is a freshman at Duke.  He plans to pursue medicine as a career. His interests lie in Detroit sports, tennis, and dancing. He is a member of the DBS Raas team on campus.  He is currently in the Trinity School of the Arts and Sciences but is considering transferring to the Pratt School of Engineering.  He hopes that the Detroit Lions may one day win the Superbowl. While waiting for this to occur, he will attend the iGEM competition and is looking forward to winning an award.
Sahil Prasada is a freshman at Duke.  He plans to pursue medicine as a career. His interests lie in Detroit sports, tennis, and dancing. He is a member of the DBS Raas team on campus.  He is currently in the Trinity School of the Arts and Sciences but is considering transferring to the Pratt School of Engineering.  He hopes that the Detroit Lions may one day win the Superbowl. While waiting for this to occur, he will attend the iGEM competition and is looking forward to winning an award.
|-
|-
-
|[[Image:Quanm.jpg|250px]]
+
|[[Image:Nicholas.jpg|200px]]
-
|'''Maggie Jiayuan Quan''' <br /> jq7@duke.edu <br /> bio
+
|'''Nicholas Tang''' <br /> nicholas.tang(at)duke.edu <br />
 +
Nicholas Tang is a junior Biomedical Engineering/Electrical and Computer Engineering student. He plans to pursue his interests in computational biology and biological engineering. His hobbies include cycling, playing the violin and playing the piano. As a participant in the 2006, 2007, and 2008 Duke iGEM teams, he is excited to contribute to the progress of the synthetic biology community, and hopes to help in any way he can.
|-
|-
-
|[[Image:Nicholas.jpg|250px]]
+
|[[Image:zhup.jpg|200px]]
-
|'''Nicholas Tang''' <br /> nicholas.tang@duke.edu <br /> bio
+
|'''Peter Zhu''' <br /> peter.zhu(at)duke.edu <br /> Peter Zhu Is a freshman at Duke University and a North Carolina local. Though he's not sure yet what to do with his life, he thinks Biomedical Engineering and pre-Law is looking pretty good. When he's not busy with the routines of life, he is listening to the Billboard Top 100, playing Chopin Preludes, searching for new places to eat, playing tennis, studying poker, and gaming Starcraft/DoTA. Peter is a regular at Bail Hai Mongolian Grill, Lime and Basil Vietnamese Pho, and Five Guy's Burgers---bacon cheeseburger with all toppings of course.
-
|-
+
-
|[[Image:zhup.jpg|250px]]
+
-
|'''Peter Zhu''' <br /> peter.zhu@duke.edu <br /> Peter Zhu Is a freshman at Duke University and a North Carolina local. Though he's not sure yet what to do with his life, he thinks Biomedical Engineering and pre-Law is looking pretty good. When he's not busy with the routines of life, he is listening to the Billboard Top 100, playing Chopin Preludes, searching for new places to eat, playing tennis, studying poker, and gaming Starcraft/DoTA. Peter is a regular at Bail Hai Mongolian Grill, Lime and Basil Vietnamese Pho, and Five Guy's Burgers---bacon cheeseburger with all toppings of course.
+
|-
|-
|}
|}
 +
<html><h2>Faculty Advisors</h2></html>
 +
{|
 +
|[[Image:TianJ.jpg|100px]]
 +
|'''Dr. Jingdong Tian''' <br /> jtian(at)duke.edu <br />Department of Biomedical Engineering & IGSP
 +
|[[Image:YouL.jpg|100px]]
 +
|'''Dr. Lingchong You''' <br /> you(at)duke.edu <br /> Department of Biomedical Engineering & IGSP
 +
|[[Image:Yuanf.jpg|100px]]
 +
|'''Dr. Fan Yuan''' <br /> fyuan(at)duke.edu <br /> Department of Biomedical Engineering
 +
|}
 +
{|
 +
|[[Image:Quanm.jpg|100px]]
 +
|'''Maggie Jiayuan Quan''' <br /> jq7(at)duke.edu <br /> Graduate Student
 +
|[[Image:Rezaf.jpg|150px]]
 +
|'''Faisal Reza''' <br /> faisal.reza(at)duke.edu <br /> Graduate Student
 +
|}
 +
<html>
<html>
</div>
</div>
-
<div id="about-box">  
+
<h3>Sponsors</h3></html>
-
<h1> About Duke University </h1></div>
+
 
 +
----
 +
 
 +
{| class="wikitable" border="0" style="margin:auto"
 +
|-
 +
|[[Image:LORDlogo.gif|150px]]
 +
|[[Image:Bmeheader.gif|650px]]
 +
|[[Image:igsp.png|100px]]
 +
|}
 +
<html>
</html>
</html>

Latest revision as of 04:00, 22 October 2009

One-Step Construction of a Bioplastic Production Pathway in E. coli


We, the determined and motivated Duke University iGEM 2009 team of 6 undergraduate students and 3 graduate students, have developed a practical and powerful method to deliver on the promise of sustainable green synthetic biology!

Due to the high costs and inefficiency of the gene and pathway cloning process, we invented a new procedure which lowers costs and increases efficiency. This method, Circular Polymerase Extension Cloning (CPEC), saves time as well, since it does not involve ligation or restriction enzymes. See publication of our method here.

We have applied our method to the production of biologically derived plastics. The high costs of the current method of producing biodegradable plastics in an environmentally sound manner has hindered its widespread use. However, we have discovered a more efficient pathway to produce these biodegradable plastics.




Why do we deserve GOLD?

  • We had a Great summer working on our project
  • We submitted 2 biobricks: BBa_K282001 and BBa_K282000
  • We Created and implemented with great success a more accurate, flexible and efficient method of splicing multi-component biobricks systems into plasmid vectors.
  • and...

*We were also proud to help Team Valencia with their survey.

Abstract

A convenient ligation-free, sequence-independent one-step plasmid assembly and cloning method is developed [Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4(7): e6441]. This strategy, Circular Polymerase Assembly Cloning (CPEC), relies solely on polymerase extension to assemble and clone multiple fragments into any vector. Using this method, we are able to quickly assemble a metabolic pathway consisting of multiple enzymes and regulatory elements for the production of a biocompatible as well as biodegradable plastic polymer in E. coli.

What is CPEC?

Circular Polymerase Extension Cloning (CPEC) is a much simplified sequence-independent cloning technology based entirely on the polymerase extension mechanism. This method extends overlapping regions between the insert and vector fragments to form a complete circular plasmid (Figure 1a). The basic principle of this strategy is that, after denaturation, both single-stranded vector and inserts overlap with each other by end sequences and extend by using the other as the primer in a typical PCR reaction. After complete circular plasmids with one nick in each strand are formed, the relaxed plasmids are transformed to competent cells and sealed as closed plasmids. CPEC strategy is not only suitable for the cloning of an individual gene but also for gene libraries, combinatorial libraries and multi-fragment plasmid assembling. With its simplicity, cost-effectiveness and high transformation efficiency, CPEC has become the most convenient, economical and accurate cloning method at the present day, especially for cloning applications with high complexity.


Figure 1a. Biobrick spliced into vector using CPEC
Figure 1b. Multicomponent Biobrick system spliced into vector simulatenously using CPEC

One-Step Construction of a Bioplastic Production Pathway using CPEC

We applied CPEC in the construction of a multi-component pathways in a single reaction. The proposed multi-way cloning mechanism is shown in Figure 1b. First, we constructed the biobrick parts containing genes involved in this pathway. Then we used multi-way CPEC for construction of a metabolic pathway for synthesizing a biodegradable plastic, poly(3HB-co-4HB) in E. coli.

Biodegradable Plastic Synthesis Pathway in E. coli.

Figure 2. PHA granule production in bacteria

Recently, polyhydroxyalkanoates (PHA) have received a lot of attention for their potential use as bioplastic. They are naturally occurring biopolymers – under low nitrogen, phosphorus, or oxygen conditions, many bacteria produce protein- and lipid-coated granules of PHA for energy storage, and these granules can then in turn be harvested in large quantities as bioplastics (Fig. 2). Bioplastics not only help reduce the reliance on petroleum-based plastics, but also have a wide range of applications in the agricultural and medical fields because they are biodegradable and biocompatible. The most common PHA is poly(3-hydroxybutyrate), or poly(3HB), which is produced by the bacteria Cupriavidus necator. Three genes – phaA, phaB, and phaC – make up the phaCAB operon that synthesizes the enzymes required for formation of poly(3HB), which is not elastic enough for general plastic applications. But when phaCAB is expressed with cat2, a succinate degradation gene from Clostridium kluyveri that produces the 4HB monomer, the copolymer poly(3-hyroxybutyrate-co-4-hydroxybutyrate), or poly(3HB-co-4HB), is produced (Figure 3). Poly(3HB-co-4HB) is more elastic and thus has potential to be used in a variety of applications.

However, bioplastic production is currently too costly to be used widely. For example, poly(3HB-co-4HB) pathways that have been developed so far produce copolymers with very low concentrations of 4HB, the monomer that gives the copolymer its elastic properties. This could be because the enzyme that catalyzes PHA formation, PHA synthase, has higher affinity for the 3HB-CoA substrate (100%) than the 4HB-CoA substrate (0.03%).

Figure 3. Pathway for poly(3HB-co-4HB) synthesis [Madison and Huisman, 1999]

In addition, the 3HB pathway uses acetyl-CoA, an input to the citric acid cycle, as a precursor, but cat2 uses succinyl- CoA, an intermediate in the cycle. Since more processing is required to obtain the 4HB cursor, the pathway may naturally favor production of the 3HB polymer, resulting in higher 3HB composition in the final copolymer.

Thus, this project aims to develop a more efficient biopathway for poly(3HB-co-4HB) while increasing the 4HB monomer composition predictably. To achieve this goal, we designed vectors with various combinations of the phaCAB operon and the cat2 gene so that genetic manipulations can be made to the genes for PHA synthase and precursor processing to increase bioplastic production and quality.

Figure 4. The reddish tint present on some colonies indicates the presence of PHA granules in the bacteria

3HB genetic pathway

The phaCAB operon genomic DNA from Cupriavidus necator was cloned into PCR Blunt II-TOPO vector as the backbone and the cloning product was transformed into bacteria cells which were then plated on kanamycin (50 μg/ml) plates. Successful insertion of phaCAB was shown by Nile Red fluorescence in colonies (Figure 4).

4HB genetic pathway

The cat2 gene was inserted into the pSOS vector to obtain the pSOS-cat2 construct. To obtain 4HB production, though, the phaC gene must also be expressed. The 4HB precursor γ-butyrolactone (GBL) is required for 4HB production in this pathway, as the 4HB precursor does not naturally exist in E. coli.


pASK vector constructs

The PCR-Blunt II-phaCAB and pSOS-cat2 plasmids described above were then used as templates to create seven different vector constructs in pASK (Tables 1 and 2). The first six vector constructs were created by PCR amplification of the insert, restriction digestion of the vector and insert, ligation of the fragments, and finally transformation into E. coli. The seventh vector (pASKphaAB-term-pLZCat2phaC) was created using CPEC cloning and transformation into E. coli (Figure 5). All the constructs were sequenced to ensure that the constructs were correct. Aslo genotyping was done for the final construct 7 (Figure 6).

Figure 5. Construct 7.
Figure 6. (Upper left) Gel electrophoresis analysis of the final assembly product after a 20-cycle CPEC. 5 ml of the reaction was separated on a 0.8% agarose gel and visualized after ethidium bromide staining. The full-length plasmid was 8360 bp. (Upper right) Gel electrophoresis analysis of the multi-way CPEC reaction. 5 ml was taken out of the reaction after 2, 5, and 10 cycles and separated on a 0.8% agarose gel (lanes 1, 2 and 3). The starting lengths of the four fragments were 3280, 2959, 2040, and 171 bp, respectively. The 171-bp band was not visible. (Bottom) Restriction mapping of the isolated plasmids derived from the CPEC reaction. Plasmid DNA from five independent colonies (I-V) were digested with BamHI (lane 2, 8.4 kb), BamHI-XhoI (lane 3, 6.6 kb and 1.8 kb), and NdeI (lane 4, 5.4 kb and 3 kb). Purified plasmids not subjected to restriction digestion are shown in lane 1. The molecular weight marker used in this figure was NEB 1 kb DNA ladder.

Screening Experiments

Two different experiments were conducted to investigate (1) the optimal anhydrotetracylcine level for inducing expression of bioplastic genes and (2) Nile Red as a screening technique for polymer production. To determine the optimal anydrotetracycline levels for phaCAB and cat2 gene expression, agar plates were prepared with 0, 5, 50, 150, and 200 ng/ml of anyhdrotetracyline, and Construct 7 E. coli were grown on them overnight at 37°C. The number of colonies on each plate was then compared the following day. To test Nile Red as a screening technique to detect PHA production, agar plates were prepared with various levels of Nile Red (0, 0.2, 0.5,1.0 μg/ml). Bacteria that were known to produce PHA (positive control) and negative control bacteria were then plated and grown overnight at 37°C. After sitting on the benchtop for a few days, they were examined under equal levels of UV lighting.

Figure.7. Precipitated product from pASK-phaC-no tag (2 tubes on left) and negative control pASK (2 tubes on right) harvesting.

Polymer harvesting

To obtain in vivo PHA polymer, E. coli containing the desired construct were grown in 3 mL of LB broth overnight, of which 1 mL was then cultured in 50 mL of LB broth and grown at 30oC until the cells reached an optical density of .7 A (600 nm). Glucose was added to a final concentration of 1%, and the cells were harvested after 48 hour of incubation at 37°C. To harvest, the cells were centrifuged at 3500 rpm and suspended in saline (0.0067 M phosphate buffer) solution. The cells were re-centrifuged to obtain a washed pellet, freezethawed in dry ice, and then freeze-dried by lyophilization. The pellet was shaken overnight at 37°C in chloroform, after which bacterial cell waste was filtered out using Whatman paper and a vacuum flask. The polymer was then precipitated from the filtered solution by adding 10 volumes of ethanol, centrifuged, and then re-dissolved in chloroform. Finally, 10 volumes of methanol was used to re-precipitate the polymer before centrifuging to obtain a purified polymer pellet. A promising sign was that there was much more white powder precipitate in the experimental samples than the negative control samples after harvesting (Figure 7).

1H NMR Spectroscopy

To characterize harvested polymers, 300 MHz 1H NMR spectroscopy was used. The NMR sample was prepared by re-dissolving 20 mg of each polymer sample in 600 μl of deuterochloroform in an NMR tube. Attempts were made to harvest and identify the 3HB, 4HB, and poly(3HB-co-4HB) polymers. The first harvest attempt was made on pUC19phaCAB and then on pASK-phaCAB-no tag (Construct 1) to try and obtain 3HB polymer; the NMR results for the harvested product were compared to that of a negative control (no insert) and commercial 3HB. Several harvest attempts were also made on pSOS-cat2phaC bacteria (Construct 4) to try to confirm 4HB polymer production. Finally, bacteria transformed with pASKphaAB-termpLZCat2phaC (Construct 7) were harvested in an attempt to identify poly(3HB-co-4HB) by NMR.


Figure 8a. 300 MHZ 1H NMR for commercial 3HB
Figure 8b. 300 MHZ 1H NMR for experimental 3HB
Figure 8c. 300 MHZ 1H NMR for negative control (pASK vector)
Figure 8d. Comparison of expansions of the methane proton region in the 1H NMR spectra –commercial 3HB (left) and experimental (right).
Figure 8e. Comparison of expansions of the CH2 regions in the 1H NMR spectra – Commercial 3HB (left) and experimental (right)
The 1H NMR spectra confirmed production of 3HB in the experimental bacteria and no 3HB production in the negative control bacteria (Fig. 8a-e). Commercial 3HB (from Sigma- Aldrich) was used as a comparison control (Fig. 8a). The 3HB peaks in the experimental sample matched the commercial 3HB sample, as well as 3HB spectra found in the literatures. However, the NMR spectra for the experimental and control samples had extremely large peaks from residual methanol and ethanol left over from the harvesting procedures, a problem corrected in later NMR readings by freeze-drying at the end of the harvesting procedure. 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
Table 1. Vector designs with various phaCAB and cat2 combinations
Table 2. Restriction enzymes and details of the vector constructs

feasible economically.



Biobricks

In order to facilitate the completion of this project, several biobricks were created and used. The two foremost (the ones submitted) were phaAB and a double terminator.

phaAB


Part: BBa_K282000
Used in the creation of bioplastics. Contains both the PhaA gene and the PhaB gene. Part of the pathway PhaAB - Terminator - Cat2PhaC .

Terminator


Part: BBa_K282001
Based off of Part:BBa_B0014. Comprised of forward terminator Part:BBa_B0010 and reverse terminator Part:BBa_B0012.

Contents

Calendar

Protocols

CPEC Cloning

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Materials

  • Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
  • Thermocycler

Preparation

5x Phusion HF Buffer    4 ul
10 mM dNTPs	        0.4 ul	
Vector		        50 ng/1kb	
Insert		        x ng*	
Phusion DNA Polymerase  0.2 ul
-------------------------------
H2O		        to 20 ul

*The amount of insert is determined so that the molar ratio for vector and insert is 1 to 2.

Procedures

98°C                  30sec
  10X
     98°C             10 sec
     Annealing**      30 sec
     72°C             x sec***
72°C                  5min
4°C                   hold

** Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.
***The extension time is usually calculated according to the shortest piece with 15 sec /kb if the cloning is not complicated. For example, if there is only one insert and is shorter than the vector, say, 600 bp, then I will use 15 sec for extension. Refer to the published paper for detailed information.

DNA Purification

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Materials :

  • E.Z.N.A Gel Purification Kit (Omega Bio-Tek, Cat No. D2500-02 )
  • Water bath equilibrated to 55-65C
  • Microcentrifuge capable of at least 10,000 x g
  • Nuclease-free 1.5 ml centrifuge bottles
  • Absolute (95%-100%) ethanol
  • Protective eye-wear
  • Isopropanol (for fragments < 500 bp only)

Protocol:

  1. Perform agarose gel electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended, however, that fresh TAE buffer or TBE buffer be used as running buffer. Do not re-use running buffer as its pH will increase and reduce yields.
  2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean scalpel.
  3. Determine the approximate volume of the gel slice by weighing it in a clean 1.5 ml microfuge tube. Assuming a density of 1 g/ml of gel, the volume of gel is derived as follows: A gel slice of mass 0.3 g will have a volume of 0.3 ml. Add equal volume of Binding Buffer (XP2). Incubate the mixture at 55C-60C for 7 min or until the gel has completely melted. Mix by shaking or inverting the tube every 2-3 minutes. Centrifuge the tube briefly to collect all the liquid to the bottom of the tube.

Note: For DNA fragment less than 500bp, add 1 sample volume of isopropanol after the addition of Binding Buffer (XP2).

  1. Apply up to 700 ul of the DNA/agarose solution to a HiBind® DNA spin column assembled in a clean 2 ml collection tube (provided) and centrifuge in a microcentrifuge at 8,000-10,000 x g for 1 min at room temperature. Discard the liquid. Re-use the collection tube in Steps 5-8. For volumes greater than 700 ul, load the column and centrifuge successively, 700 ul at a time. Each HiBind® spin-column has a total capacity of 25-30 ug DNA.
  2. Discard liquid and add 300ul Binding Buffer. Centrifuge at 10,000 x g for 1 minutes.
  3. Add 700 ul of SPW Buffer diluted with absolute ethanol into the column and wait 2-3 minutes. Centrifuge at 10,000 x g for 1 min at room temperature to wash the sample.
  4. Discard liquid and repeat step 6 with another 700 ul SPW Buffer.
  5. Discard liquid and, re-using the collection tube, centrifuge the empty column for 1 min at maxi speed (>13,000 x g) to dry the column matrix. This drying step is critical for good DNA yields.
  6. Place column into a clean 1.5 ml microcentrifuge tube (not provided). Add 30-50 ul depending on desired concentration of final product) Elution Buffer (or sterile deionized water) directly to the center of the column matrix, then incubate for 1 minute. Centrifuge 1 min at maxi speed (>13,000 x g) to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lo

PCA (Polymerase Cycle Assembly)

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Materials

  • Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
  • Thermocycler

Preparation

5x Phusion HF Buffer 5 ul
10 mM dNTPs 0.5 ul
Oligo mixture 125 ng /250 ng /500 ng /
Phusion DNA Polymerase 0.25 ul
--------------------- --------
H2O to 25 ul


Procedures

98°C              30sec 
  40X
      98°C        7 sec
      70-50°C     slow ramp, 0.1°C/sec
      50°C        30 sec 
      72°C        15 sec /kb
72°C              5 min
4°C               hold

PCR Product Clean-up for DNA Sequencing

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Materials:

  • ExoSAP-IT® (usb, Cat. No. 78200)
  • Thermocycler

Protocol:

  1. Remove ExoSAP-IT from -20°C freezer and keep on ice throughout this procedure.
  2. Mix 5 μl of a post-PCR reaction product with 2 μl of ExoSAP-IT for a combined 7 μl reaction volume.
  3. Incubate at 37°C for 15 min to degrade remaining primers and nucleotides.
  4. Incubate at 80°C for 15 min to inactivate ExoSAP-IT.
  5. The PCR product is now ready for use in DNA sequencing etc.

PCR

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Materials

  • Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
  • Thermocycler

Preperation

5x Phusion HF Buffer 10 ul
10 mM dNTPs 1 ul
DNA template 1 pg – 10 ng
Forward primer (10 uM) 2.5 ul
Reverse primer (10 uM) 2.5 ul
Phusion DNA Polymerase 0.5 ul
-------------------- -----
H2O to 50 ul


Procedure

98°C           30sec 
 30X
    98°C       10 sec
    Annealing* 30 sec
    72°C       15 sec per 1 kb
72°C           5min
4°C            hold


* Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.

Single Colony PCR

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Materials

  • Taq DNA Polymerase with Standard Taq Buffer (NEB, Cat. No. M0273)
  • 10 mM dNTP Mix (NEB, Cat. No. N0447)
  • Thermocycler

Preperation

10x Standard Taq Buffer 2 ul
10 mM dNTPs 0.4 ul
Bacteria culture* 1 ul
Forward primer (10 uM) 1 ul
Reverse primer (10 uM) 1 ul
Taq DNA Polymerase 0.2 ul
----------------- -------
H2O to 20 ul

*Bacteria culture refers to E. coli cultured in LB solution overnight.

Procedures

94°C              5 min 
 25X
     94°C         15 sec
     Annealing**  30 sec
     72°C         1 min per kb
72°C              5min
4°C               hold

**Anneal at Tm which is calculated with salt-adjusted method.

Transformation

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Materials:

  • GC5 Chemical Competent Cells (Genesee Scientific, Cat. No. 42-653)
  • SOC Medium (Sigma, Cat. No. S1797)
  • LB Agar (Sigma, Cat. No. L3027)
  • Petri Dishes (VWR, Cat. No. SC25373-187)
  • Cell Spreader (VWR, Cat. No. 89042-018)
  • 37°C incubator
  • 37°C shaker
  • water bath

Protocol:

1. Thaw 1 tube of competent cells on ice;

2. Add 3 ul of cloning product or 1-50 ng of plasmid into competent cells while stirring gently;

3. Keep the tube covered by ice for 30min;

4. Heat-shock the competent cells in water bath for 45 sec at 42°C;

5. Put the tube on ice for 2 minutes;

6. Add 450 ul of SOC medium and then put it in a 37°C shaker for 1 hour;

7. Dilute and spread an appropriate amount on an LB agar plate with the appropriate antibiotics;

8. Place the plate up-side-down in 37°C incubator for 16-18 hours (overnight).

NMR

  1. Login and start NMR program.
  2. Click Acqi. Load sample. Make sure spin is on and lock is off.
  3. Go to lock. Decrease number of sine waves to 1 to obtain step function. Make sure Zo is 1100, lockpower is below 30, lockgain is 36, lockphase is 352, and spin is 20.
  4. Go to shim. Increase lock level.
  5. Click main menu. Click set up. Click H1CDCl3. Type ‘nt=64’ (number of scans), ‘ss=2’ (dummy scans), ‘go’, ‘lb=0.2’ (line broadening).
  6. Viewing: Type ‘wft’, ‘dscale’, ‘dfp’ to display peak frequency.
  7. Zooming in: Click display. Go to interactive. Type ‘cr=8p, delta=8p’ to set width of zoom. Use cursor to set boundaries. Click to cut.
  8. Printing: Type “pL pscale(0) pltext ppf page’ to print spectra with text and peak frequencies.

Lab Safety

None of our project ideas raised any safety issues whether it be researcher, environmental, or public safety. Duke has an in-house review board for projects and they did not raise any flags regarding our project. The biobrick parts created also do not pose any safety issues.


Not Including: Andrew Ang and Kevin Chien


Students

Anga.jpg Andrew Ang
andrew.ang(at)duke.edu
Andrew Ang is a freshman at Duke, majoring in Biomedical Engineering. Apart from class and iGEM, he is involved in the Jazz Ensembles program and Asian Students Association at Duke. His hobbies include piano, saxophone, tennis, and squash. He is interested in molecular and synthetic biology, biomolecular engineering and medical research. He has previously worked as part of the MIT 2008 team, and he is excited to participate in iGEM again this year, and many more years to come.
Chienk.jpg Kevin Chien
kevin.chien(at)duke.edu

Kevin Chien is a freshman at duke, majoring in Biomedical Engineering. He wants to explore his options in many different careers. He occasionally plays Club Frisbee and is interested in many different areas of knowledge, ranging from Astrophysics to Public Policy (and even sometimes History). He worked on this iGEM team last year as well and enjoyed his stay in Boston(hopes to have a good one this year too!).

Fuy.jpg Yaoyao Fu
yf21(at)duke.edu

Yaoyao Fu is a master's student in Biomedical Engineering. She is interested in molecular synthetic biology. Her hobbies are traveling and sports.

Faith.jpg Faith Kung
fk8(at)duke.edu

Faith Kung is a senior at Duke majoring in Biomedical Engineering with minors in Music and Biology. She enjoys working in a lab. Besides academics, her hobbies include arts and crafts, dance, and figure skating. Also, she is actively involved in the IV Christian Fellowship. Faith is applying to PhD programs in Biomedical Science and hopes to pursue a career in scientific research and education. She is excited about attending the iGEM competition this year.

PrasadaS.jpg Sahil Prasada
sahil.prasada(at)duke.edu

Sahil Prasada is a freshman at Duke. He plans to pursue medicine as a career. His interests lie in Detroit sports, tennis, and dancing. He is a member of the DBS Raas team on campus. He is currently in the Trinity School of the Arts and Sciences but is considering transferring to the Pratt School of Engineering. He hopes that the Detroit Lions may one day win the Superbowl. While waiting for this to occur, he will attend the iGEM competition and is looking forward to winning an award.

Nicholas.jpg Nicholas Tang
nicholas.tang(at)duke.edu

Nicholas Tang is a junior Biomedical Engineering/Electrical and Computer Engineering student. He plans to pursue his interests in computational biology and biological engineering. His hobbies include cycling, playing the violin and playing the piano. As a participant in the 2006, 2007, and 2008 Duke iGEM teams, he is excited to contribute to the progress of the synthetic biology community, and hopes to help in any way he can.

Zhup.jpg Peter Zhu
peter.zhu(at)duke.edu
Peter Zhu Is a freshman at Duke University and a North Carolina local. Though he's not sure yet what to do with his life, he thinks Biomedical Engineering and pre-Law is looking pretty good. When he's not busy with the routines of life, he is listening to the Billboard Top 100, playing Chopin Preludes, searching for new places to eat, playing tennis, studying poker, and gaming Starcraft/DoTA. Peter is a regular at Bail Hai Mongolian Grill, Lime and Basil Vietnamese Pho, and Five Guy's Burgers---bacon cheeseburger with all toppings of course.

Faculty Advisors

TianJ.jpg Dr. Jingdong Tian
jtian(at)duke.edu
Department of Biomedical Engineering & IGSP
YouL.jpg Dr. Lingchong You
you(at)duke.edu
Department of Biomedical Engineering & IGSP
Yuanf.jpg Dr. Fan Yuan
fyuan(at)duke.edu
Department of Biomedical Engineering
Quanm.jpg Maggie Jiayuan Quan
jq7(at)duke.edu
Graduate Student
Rezaf.jpg Faisal Reza
faisal.reza(at)duke.edu
Graduate Student

Sponsors


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