Difference between revisions of "Team:NRP-UEA-Norwich/Results/Prebiotic"

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            <h2 class="title1 f900" id="top2">RESULTS</h2>
 
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                      <li role="presentation" class="purple scroll-link" data-id="top2"><a href="#home1" aria-controls="home1" role="tab" data-toggle="tab"><span>- Glycogen branching constructs</span></a></li>
 
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<li role="presentation" class="purple scroll-link  space50" data-id="top2"><a href="#home2" aria-controls="home1" role="tab" data-toggle="tab"><span>- Glycogen acyltransferase constructs </span></a></li>
 
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                      <li role="presentation" class="purple scroll-link space50" data-id="top2"><a href="#profile" aria-controls="profile" role="tab" data-toggle="tab"><span>- Glycogen content (1)</span></a></li>
 
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                      <li role="presentation" class="purple scroll-link" data-id="top2"><a href="#messages" aria-controls="messages" role="tab" data-toggle="tab"><span>- Glycogen content (2)</span></a></li>
 
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                      <li role="presentation" class="purple scroll-link" data-id="top2"><a href="#setting4" aria-controls="setting4" role="tab" data-toggle="tab"><span>- Cloning of plant constructs</span></a></li>
 
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                      <li role="presentation" class="purple scroll-link space50" data-id="top2"><a href="#setting5" aria-controls="setting5" role="tab" data-toggle="tab"><span>- Chloroplast localisation</span></a></li>
 
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<li role="presentation" class="purple scroll-link" data-id="top2"><a href="#setting6" aria-controls="content4" role="tab" data-toggle="tab"><span>- Starch content</span></a></li>
 
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<li role="presentation" class="aqua scroll-link" data-id="top2"><a href="#pre1" aria-controls="pre1" role="tab" data-toggle="tab"><span>- Butyrate Pathway I</span></a></li>
 
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<li role="presentation" class="aqua scroll-link" data-id="top2"><a href="#pre2" aria-controls="pre2" role="tab" data-toggle="tab"><span>- Butyrate Pathway II</span></a></li>
 
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<li role="presentation" class="aqua scroll-link" data-id="top2"><a href="#pre3" aria-controls="pre3" role="tab" data-toggle="tab"><span>- Production of butyryl coA in <i>E. coli</i></span></a></li>
 
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<li role="presentation" class="aqua scroll-link space50" data-id="top2"><a href="#pre4" aria-controls="pre4" role="tab" data-toggle="tab"><span>- Production of butyryl coA in <i>E. coli</i></span></a></li>
 
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                                  <h2 class="title1">Making constructs to express inducible branching and debranching enzymes in <i>E. coli</i></h2>
 
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<h4 class="title2">AIM:</h4>
 
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                        <p class="space30">We aimed to ligate the <i>E. coli</i> glycogen metabolic genes <a href="http://parts.igem.org/Part:BBa_K1618025"style="color:#002bb8;">GlgX</a> and <a href="http://parts.igem.org/Part:BBa_K1618000"style="color:#002bb8;">GlgB</a> into the standard pSB1C3 plasmid vector with and without an <a href="http://parts.igem.org/Part:BBa_J04500"style="color:#002bb8;">IPTG-inducible promoter</a>. GlgX is a glycogen debranching enzyme which cleaves &alpha;-1,6 glycosidic linkages in glycogen to remove branches. GlgB is a glycogen branching enzyme which cleaves &alpha;-1,4 glycosidic linkages and re-anneals the cleaved linkage back onto the main chain through an &alpha;-1,6 linkage, therefore creating a branch. Creating parts with the promoter allows us to investigate the effect of the expression of these enzymes on <i>E. coli</i> glycogen in an inducible system. Basic parts with just the enzyme coding sequences were also generated so that they can be used to build other composite parts in the future.</p>
 
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<h4 class="title2">METHOD:</h4>
 
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                        <p class="space20">To achieve the cloning, we digested the various DNA components (<a href="http://parts.igem.org/Part:BBa_K1618025"style="color:#002bb8;">GlgX</a> or <a href="http://parts.igem.org/Part:BBa_K1618000"style="color:#002bb8;">GlgB</a> sequence, pSB1C3 and <a href="http://parts.igem.org/Part:BBa_J04500"style="color:#002bb8;">IPTG-inducible promoter</a>) according to the <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Restriction Digest Protocol for BioBrick" style = "color: #002bb8;">restriction digest protocol</a> with the relevant restriction enzymes to create compatible ends. The digestions were then run on an <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Agarose Gel Electrophoresis Protocol" style = "color: #002bb8;">agarose gel</a> and <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#QIAGEN Gel Extraction Protocol" style = "color: #002bb8;">gel extracted</a> to yield the digested DNA components. These were <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Ligation Protocol" style = "color: #002bb8;">ligated</a> and <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Heat-Shock Transformation Protocol" style = "color: #002bb8;">transformed</a> into competent <i>E. coli</i> DH5&alpha; cells. A <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Colony PCR Protocol" style = "color: #002bb8;">colony PCR</a> was undertaken and liquid cultures of colonies with the correctly-sized inserts were prepared. The plasmids were purified by <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#QIAGEN Plasmid Prep Protocol" style = "color: #002bb8;">plasmid prep</a> and sent for sequencing. After sequencing a mutation in the terminator codon of the GlgX sequence was discovered and this was subsequently corrected with <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Mutagenesis PCR Protocol" style = "color: #002bb8;">mutagenesis PCR </a> to give the correct sequence.</p>
 
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<h4 class="title2">RESULTS:</h4>
 
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<p class="space20">The resulting plasmids were <a href="http://parts.igem.org/Part:BBa_K1618026"style="color:#002bb8;">GlgX with IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618025"style="color:#002bb8;">GlgX without IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618022"style="color:#002bb8;">GlgB with IPTG-inducible promoter</a>, and <a href="http://parts.igem.org/Part:BBa_K1618000"style="color:#002bb8;">GlgB without IPTG-inducible promoter</a>.</p>
 
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<img src="https://static.igem.org/mediawiki/2015/e/e7/NRP-Mark-Results1Image1.png" href="https://static.igem.org/mediawiki/2015/e/e7/NRP-Mark-Results1Image1.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: Plasmid diagrams of pSB1C3 containing an <a href="http://parts.igem.org/Part:BBa_J04500"style="color:#002bb8;">IPTG-inducible promoter</a> (lacI), RBS and GlgX (left, <a href="http://parts.igem.org/Part:BBa_K1618026"style="color:#002bb8;">BBa_K1618026</a>) and pSB1C3 containing only the GlgX genetic sequence (right, <a href="http://parts.igem.org/Part:BBa_K1618025"style="color:#002bb8;">BBa_K1618025</a>).</p>
 
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<img src="https://static.igem.org/mediawiki/2015/5/54/NRP-Mark-Results1Image2.png" href="https://static.igem.org/mediawiki/2015/5/54/NRP-Mark-Results1Image2.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: Agarose gel electrophoresis image after mutagenesis PCR. Colony PCR gel? The GlgX insert is 2019 base pairs long and comparing the bands on the gel to the 2-log ladder, the GlgX bands reside just above the 2000 marker, therefore suggesting GlgX as the insert. Genetic sequencing confirmed the GlgX genetic sequence in the pSB1C3 vector was correct. Lane key or label?</p>
 
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<img src="https://static.igem.org/mediawiki/2015/e/e1/NRP-Mark-Results1Image3.png" href="https://static.igem.org/mediawiki/2015/e/e1/NRP-Mark-Results1Image3.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 3</b>: Plasmid diagram of pSB1C3 containing the <a href="http://parts.igem.org/Part:BBa_J04500"style="color:#002bb8;">IPTG-inducible promoter</a> (lacI) RBS and GlgB (left, <a href="http://parts.igem.org/Part:BBa_K1618022"style="color:#002bb8;">BBa_K1618022</a>) and pSB1C3 containing only the GlgB genetic sequence (right, <a href="http://parts.igem.org/Part:BBa_K1618000"style="color:#002bb8;">BBa_K1618000</a>).</p>
 
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<img src="https://static.igem.org/mediawiki/2015/3/3d/NRP-Mark-Results1Image4.png" href="https://static.igem.org/mediawiki/2015/3/3d/NRP-Mark-Results1Image4.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 4</b>: Agarose gel electrophoresis image for correct GlgB cloning. The GlgB insert is 2231 base pairs long and comparing the bands on the gel to the 2-log ladder, the GlgB bands reside just above the 2000 marker, therefore suggesting GlgB as the insert. Genetic sequencing confirmed the GlgB genetic sequence in the pSB1C3 vector was correct.</p>
 
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                                  <h2 class="title1">Making constructs to express putative α-glucan acyltransferases in E. coli</h2>
 
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<h4 class="title2">AIM:</h4>
 
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                        <p class="space30">We aimed to ligate four putative α-glucan acyltransferase genetic sequences: <a href="http://parts.igem.org/Part:BBa_K1618001"style="color:#002bb8;">MAO</a>, <a href="http://parts.igem.org/Part:BBa_K1618002"style="color:#002bb8;">Rv3030</a>, <a href="http://parts.igem.org/Part:BBa_K1618003"style="color:#002bb8;">Rv3034c</a>, and <a href="http://parts.igem.org/Part:BBa_K1618004"style="color:#002bb8;">Rv3037c</a> into the standard pSB1C3 plasmid vector with and without an <a href="http://parts.igem.org/Part:BBa_J04500"style="color:#002bb8;">IPTG-inducible promoter</a>. These acyltransferases are considered to be able to transfer acyl groups from acyl-CoA coenzymes onto α-glucans such as glycogen and starch.</p>
 
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<h4 class="title2">METHOD:</h4>
 
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                        <p class="space20">To achieve the cloning, we digested the various DNA components (putative acyltransferase sequences, pSB1C3 and <a href="http://parts.igem.org/Part:BBa_J04500"style="color:#002bb8;">IPTG-inducible promoter</a>) according to the <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Restriction Digest Protocol for BioBrick" style = "color: #002bb8;">restriction digest protocol</a> with the relevant restriction enzymes to create compatible ends. The digestions were then run on an <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Agarose Gel Electrophoresis Protocol" style = "color: #002bb8;">agarose gel</a> and <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#QIAGEN Gel Extraction Protocol" style = "color: #002bb8;">gel extraction</a> to yield the digested DNA components. These were <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Ligation Protocol" style = "color: #002bb8;">ligated</a> and <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Heat-Shock Transformation Protocol" style = "color: #002bb8;">transformed</a> into competent  E. coli DH5α cells. A <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Colony PCR Protocol" style = "color: #002bb8;">colony PCR</a> was undertaken and liquid cultures of colonies with the correctly-sized inserts were prepared. The plasmids were purified by <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#QIAGEN Plasmid Prep Protocol" style = "color: #002bb8;">plasmid prep</a> and sent for sequencing</p>
 
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<h4 class="title2">RESULTS:</h4>
 
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<p class="space20">The resulting plasmids were <a href="http://parts.igem.org/Part:BBa_K1618005"style="color:#002bb8;">MAO with IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618001"style="color:#002bb8;">MAO without IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618007"style="color:#002bb8;">Rv3030 with IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618002"style="color:#002bb8;">Rv3030 without IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618008"style="color:#002bb8;">Rv3034c with IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618003"style="color:#002bb8;">Rv3034c without IPTG-inducible promoter</a>, <a href="http://parts.igem.org/Part:BBa_K1618009"style="color:#002bb8;">Rv3037c with IPTG-inducible promoter</a>, and <a href="http://parts.igem.org/Part:BBa_K1618004"style="color:#002bb8;">Rv3037c without IPTG-inducible promoter</a>.</p>
 
−
<p class="space20"></p>
 
−
<img src="https://static.igem.org/mediawiki/2015/0/08/NRP-Mark-Results2Image1.png" href="https://static.igem.org/mediawiki/2015/0/08/NRP-Mark-Results2Image1.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 1</b>: Plasmid diagrams of pSB1C3 containing the <a href="http://parts.igem.org/Part:BBa_J04500"style="color:#002bb8;">IPTG-inducible promoter</a> (lacI), RBS and each of the four putative acyltransferases, MAO (<a href="http://parts.igem.org/Part:BBa_K1618005"style="color:#002bb8;">BBa_K1618005</a>), Rv3030 (<a href="http://parts.igem.org/Part:BBa_K1618007"style="color:#002bb8;">BBa_K1618007</a>), Rv3034c (<a href="http://parts.igem.org/Part:BBa_K1618008"style="color:#002bb8;">BBa_K1618008</a>) and Rv3037c (<a href="http://parts.igem.org/Part:BBa_K1618009"style="color:#002bb8;">BBa_K1618009</a>).</p>
 
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−
<p class="space20"></p>
 
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−
<img src="https://static.igem.org/mediawiki/2015/4/4a/NRP-Mark-Results2Image2.png" href="https://static.igem.org/mediawiki/2015/4/4a/NRP-Mark-Results2Image2.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 2</b>: Plasmid diagrams of pSB1C3 with each of the four acyltransferases, MAO (<a href="http://parts.igem.org/Part:BBa_K1618001"style="color:#002bb8;">BBa_K1618001</a>), Rv3030 (<a href="http://parts.igem.org/Part:BBa_K1618002"style="color:#002bb8;">BBa_K1618002</a>), Rv3034c (<a href="http://parts.igem.org/Part:BBa_K1618003"style="color:#002bb8;">BBa_K1618003</a>) and Rv3037c (<a href="http://parts.igem.org/Part:BBa_K1618004"style="color:#002bb8;">BBa_K1618004</a>).</p>
 
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−
<div class="space20"></div>
 
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−
<img src="https://static.igem.org/mediawiki/2015/5/5f/NRP-Mark-Results2Image3.png" href="https://static.igem.org/mediawiki/2015/5/5f/NRP-Mark-Results2Image3.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 3</b>: Agarose gel electrophoresis image of MAO (600 bp), Rv3030 (850), and Rv3034c (950) at the correct heights relative to the 2 log ladder. With promoter? The first non-ladder well contains an RFP control. Lane key or label? In this case no bands could be seen in lanes 16-19 for Rv3037c. Genetic sequencing confirmed that MAO, Rv3030, Rv3034c had successfully been inserted into the pSB1C3 vector, with and without promoter.</p>
 
−
<p class="space20"></p>
 
−
<img src="https://static.igem.org/mediawiki/2015/7/72/NRP-Mark-Results2Image4.png" href="https://static.igem.org/mediawiki/2015/7/72/NRP-Mark-Results2Image4.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 4</b>: Agarose gel electrophoresis image of Rv3037c (1110) at the correct height relative to the 2-log ladder. Genetic sequencing was successful for Rv3037c with and without promoter. Lane key or label? </p>
 
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                                  <h2 class="title1">Determining if expression of glycogen branching and debranching enzymes affect <i>E. coli</i> glycogen content</h2>
 
−
<h4 class="title2">AIM:</h4>
 
−
                        <p class="space30">To analyse the effect of GlgX (glycogen debranching enzyme) and GlgB (glycogen branching enzyme) expression on glycogen content in <i>E. coli</i>. We initially set out to investigate by looking at the staining of whole-cell extracts with Lugol’s solution, a method which a previous iGEM team had used. The iodine in the Lugol’s solution intercalates in the glucan chains to give a coloured complex. Comparison between the colours observed can give an indication of the amount and branching structure of the glycogen within the sample<sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">1</a></sub>. </p>
 
−
<h4 class="title2">METHOD:</h4>
 
−
                        <p class="space20">Competent <i>E. coli</i> were transformed with GlgX and GlgB under the control of an inducible promoter and firstly grown in 10 mL LB media overnight at 37 °C with shaking. Each culture was used to inoculate 2 x 10 mL of fresh media, grown to an OD of approximately 0.6 and then IPTG was added to one of each duplicate culture and the cultures continued to grow, with samples taken after 1 hour, 3 hours and overnight. At each timepoint a 1 mL extract was taken, spun down to pellet the cells and this pellet was re-suspended in Lugol solution, similar to a method previously been used by the <a href="https://2008.igem.org/Team:Edinburgh/Results/Glycogen2" style = "color: #002bb8;">Edinburgh 2008 iGEM team</a>.</p>
 
−
 
−
 
−
<p class="space20">The expected colour upon Lugol addition to glycogen is a dark brown colour, however, as shown in Figures 1-5, no sample has turned this colour with the majority of the colour seen identical to the orange/yellow of Lugol itself. We therefore repeated this method with nitrogen-limited, carbon rich minimal media M9 minimal media, which had previously been shown to lead to the accumulation of bacterial glycogen<sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">2</a></sub>.</p>
 
−
 
−
<p class="space20">Cells grew very slowly under these conditions and only reached an OD of approximately 0.6 after 24 hours.</p>
 
−
 
−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20">Figures 1 -5 show images of pelleted bacterial cells from LB media, resuspended in Lugol’s solution, and in every case it was not different to Lugol solution alone. 18 hours after induction The results from high carbon:nitrogen media are shown in Figure 5, in which every sample has a darker, browner colour than Lugol itself indicating a higher glycogen presence compared to LB media. Unfortunately solid particulates remaining in the samples precluded quantifying the results by measuring the absorbance with a spectrophotometer. Whilst there was clearly more glycogen in these samples, it was not possible to distinguish colour differences between the branching and debranching enzymes or between the IPTG-induced and non-induced samples. Therefore it was decided to extract the glycogen from the bacterial cells to analyse it directly.</p>
 
−
 
−
<img src="https://static.igem.org/mediawiki/2015/1/17/NRP-Mark-Results3Image3.png" href="https://static.igem.org/mediawiki/2015/1/17/NRP-Mark-Results3Image3.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 1</b>: The pelleted bacterial cells from 1 mL overnight LB cultures with no addition of IPTG re-suspended in 100 uL Lugol’s solution</p>
 
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<p class="space20"></p>
 
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<div class="space20"></div>
 
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−
<img src="https://static.igem.org/mediawiki/2015/3/35/NRP-Mark-Results3Image4.png" href="https://static.igem.org/mediawiki/2015/3/35/NRP-Mark-Results3Image4.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: The pelleted bacterial cells from 1 mL of LB culture an hour after IPTG-addition re-suspended in 100 uL Lugol’s solution</p>
 
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<p class="space20"></p>
 
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<div class="space20"></div>
 
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<img src="https://static.igem.org/mediawiki/2015/7/7b/NRP-Mark-Results3Image5.png" href="https://static.igem.org/mediawiki/2015/7/7b/NRP-Mark-Results3Image5.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 3</b>: The pelleted bacterial cells from 1 mL of LB culture 3 hours after IPTG-addition re-suspended in 100 uL Lugol’s solution</p>
 
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<p class="space20"></p>
 
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<div class="space20"></div>
 
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<img src="https://static.igem.org/mediawiki/2015/9/96/NRP-Mark-Results3Image6.png" href="https://static.igem.org/mediawiki/2015/9/96/NRP-Mark-Results3Image6.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 4</b>: The pelleted bacterial cells from 1 mL of LB culture 18 hours after IPTG-addition, re-suspended in 100 uL Lugol’s solution. The lighter colours observed are due to solid particulates in the media.</p>
 
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<p class="space20"></p>
 
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<div class="space20"></div>
 
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<img src="https://static.igem.org/mediawiki/2015/d/df/NRP-Mark-Results3Image7.png" href="https://static.igem.org/mediawiki/2015/d/df/NRP-Mark-Results3Image7.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 5</b>: Pelleted bacterial cells of the branching enzymes grown overnight in M9 minimal media with or without IPTG, and resuspended in Lugol’s solution.</p>
 
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<div class="space20"></div>
 
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<div class="space20"></div>
 
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<h2 class="title1" id="ref">References</h2>
 
−
    <p><a name="1"></a>1. Dreiling C, Brown D, Casale L, Kelly L. Muscle glycogen: Comparison of iodine binding and enzyme digestion assays and application to meat samples. Meat Science. 1987;20(3):167-177. </p>
 
−
<p><a name="2"></a>2. ANTOINE A, TEPPER B. Environmental Control of Glycogen and Lipid Content of Mycobacterium phlei. Journal of General Microbiology. 1969;55(2):217-226.</p>
 
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                                  <h2 class="title1">Determining if expression of glycogen branching and debranching enzymes affect <i>E. coli</i> glycogen content (2)</h2>
 
−
<h4 class="title2">AIM:</h4>
 
−
                        <p class="space30">To analyse the effect of GlgX (glycogen debranching enzyme) and GlgB (glycogen branching enzyme) expression on glycogen content in <i>E. coli</i>. As we were not able see any differences in whole-cell extracts decided to extract the glycogen from the bacterial cells and analyse it directly.</p>
 
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<h4 class="title2">METHOD:</h4>
 
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                        <p class="space20"></p>
 
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<p class="space20"> </p>
 
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<p class="space20"></p>
 
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<h4 class="title2">RESULTS:</h4>
 
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<p class="space20"></p>
 
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−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: </p>
 
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<div class="space20"></div>
 
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<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: </p>
 
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<p class="space20"></p>
 
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                                  <h2 class="title1">Title</h2>
 
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<h4 class="title2">AIM:</h4>
 
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                        <p class="space30"></p>
 
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<h4 class="title2">METHOD:</h4>
 
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                        <p class="space20"></p>
 
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<p class="space20"> </p>
 
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<p class="space20"></p>
 
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−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20"></p>
 
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−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 1</b>: </p>
 
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<p class="space20"></p>
 
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−
<div class="space20"></div>
 
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−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: </p>
 
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<p class="space20"></p>
 
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                                  <h2 class="title1">Title</h2>
 
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<h4 class="title2">AIM:</h4>
 
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                        <p class="space30"></p>
 
−
<h4 class="title2">METHOD:</h4>
 
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                        <p class="space20"></p>
 
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−
<p class="space20"> </p>
 
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<p class="space20"></p>
 
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−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20"></p>
 
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−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: </p>
 
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<p class="space20"></p>
 
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−
<div class="space20"></div>
 
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<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: </p>
 
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<p class="space20"></p>
 
−
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                                  <h2 class="title1">Title</h2>
 
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<h4 class="title2">AIM:</h4>
 
−
                        <p class="space30"></p>
 
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<h4 class="title2">METHOD:</h4>
 
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                        <p class="space20"></p>
 
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−
<p class="space20"> </p>
 
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<p class="space20"></p>
 
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−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20"></p>
 
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−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 1</b>: </p>
 
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<p class="space20"></p>
 
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−
<div class="space20"></div>
 
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−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: </p>
 
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<p class="space20"></p>
 
−
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                    </div>
 
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                        <h2 class="title1">Making constructs to express putative acyltransferases in plant chloroplasts</h2>
 
−
                        <h4 class="title2">AIM:</h4>
 
−
 
−
                        <p class="space10">The aim of our prebiotic is to produce acylated/butrylated starch in plants. Methods to chemically acylate starch purified from plants already exist, but as they use strong chemicals and require heating they are not environmentally friendly. Using various acyltransferases, we hope to acylate starch in plants. We’ll be using a model plant, <i>Nicotinia benthamiana</i>, for initial tests because we can get results within  a few days. Later on we’d aim to make transgenic plants in a species that makes a lot of starch such as maize (corn), potatoes or wheat.</p>
 
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                      </div>
 
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                        <div class="col-md-12">
 
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<h4 class="title2">METHOD:</h4>
 
−
                            <p class="space10">We used Golden Gate Cloning  and the Plant Standard Syntax<sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">1</a></sub> to make our constructs. We used 35s promoter from Cauliflower Mosiac Virus (<a href="http://parts.igem.org/Part:BBa_K1467101"style="color:#002bb8;">BBa_K1467101</a>), to drive constitutive expression.  We made a chloroplast transit peptide (New part - <a href="http://parts.igem.org/Part:BBa_K1618028"style="color:#002bb8;">BBa_K1618028</a>) that we then used in an N-terminal translational fusion with the acyl transferases for the protein to reach the chloroplast, where starch is produced.</p>
 
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−
<img src="https://static.igem.org/mediawiki/2015/f/f5/NRP-UEA-Norwich-results-leda1.png" href="https://static.igem.org/mediawiki/2015/f/f5/NRP-UEA-Norwich-results-leda1.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
                            <p class="space20">In order to confirm that the transit peptide was functional we made a second set of constructs that had a fluorescent reporter in a C-terminal translational fusion.</p>
 
−
 
 
−
<img src="https://static.igem.org/mediawiki/2015/a/a9/NRP-UEA-Norwich-results-leda2.png" href="https://static.igem.org/mediawiki/2015/a/a9/NRP-UEA-Norwich-results-leda2.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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−
<p class="space20">To deliver the constructs to plants we needed to use <i>Agrobacterium tumefaciens</i> as a shuttle chassis. This meant parts needed to be assembled into a binary vector with origins of replication for both <i>E.coli</i> and <i>A. tumefaciens</i>. In order to submit the parts to the registry we also assembled parts into the MoCloFlipper pSB1C3 that accepts Golden Gate Parts into the pSB1C3 backbone.</p>
 
−
 
−
<p>All cloning was done according to the Golden Gate one-step Digestion-Ligation Protocol provided on our protocols page <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Ones-Step Golden Gate Digestion Ligation Protocol" style = "color: #002bb8;">here</a>.</p>
 
−
 
−
<h4 class="title2">Results:</h4>
 
−
<p class="space20"><b>Successful assembly into the binary vector.</b></p>
 
−
                            <p class="space10">Putative clones were screened by colony PCR (see Protocols for method - <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Colony PCR Protocol" style = "color: #002bb8;">here</a>) using primers that flank the insertion sites (Figure 1).
 
−
</p>
 
−
 
−
<img src="https://static.igem.org/mediawiki/2015/2/27/NRP-UEA_Leda_Cloning_Result.jpeg" href="https://static.igem.org/mediawiki/2015/2/27/NRP-UEA_Leda_Cloning_Result.jpeg" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 1</b>: The gel lanes are as follows –  1 =<i> Ladder</i>, 2&3 = <i><a href="http://parts.igem.org/Part:BBa_K1618033"style="color:#002bb8;">BBa_K1618033</a></i>, 4&5 = <i><a href="http://parts.igem.org/Part:BBa_K1618029"style="color:#002bb8;">BBa_K1618029</a></i>, 6&7 = <i> <a href="http://parts.igem.org/Part:BBa_K1618035"style="color:#002bb8;">BBa_K1618035</a></i>, 8&9 = <i><a href="http://parts.igem.org/Part:BBa_K1618031"style="color:#002bb8;">BBa_K1618031</a></i>, 10&11 = <i> <a href="http://parts.igem.org/Part:BBa_K1618036"style="color:#002bb8;">BBa_K1618036</a></i>, 12&13 = <i><a href="http://parts.igem.org/Part:BBa_K1618032"style="color:#002bb8;">BBa_K1618032</a></i>, 14&15 = <i><a href="http://parts.igem.org/Part:BBa_K1618034"style="color:#002bb8;">BBa_K1618034</a></i>, 16&17 = <i><a href="http://parts.igem.org/Part:BBa_K1618030"style="color:#002bb8;">BBa_K1618030</a></i>, 18 = ladder. The gel image above indicated that the complete transcriptional units were successfully cloned. </p>
 
−
 
−
<p class="space20">A single colony for each construct was mini-prepped and sequenced before progression to transformation into <i>A. tumefaciens</i>.</p>
 
−
<p class="space20"><b>Successful assembly into pSB1C3.</b></p>
 
−
 
−
                            <p class="space30">Putative clones were miniprepped (see Protocols for method - <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#QIAGEN Plasmid Prep Protocol" style = "color: #002bb8;">here</a>) and screened by digestion with <i>Not</i>I (Figure 2) and also with
 
−
<i>EcoR</i>I/<i>Pstl</i> (Figure 3) to confirm that cloning was successful and that our constructs had no internal BioBrick restriction sites. </p>
 
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−
<img src="https://static.igem.org/mediawiki/2015/2/2e/NRP-UEA_Leda_Not1_Digest.jpeg" href="https://static.igem.org/mediawiki/2015/2/2e/NRP-UEA_Leda_Not1_Digest.jpeg" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: The gel lanes are as follows –  1 =<i> Ladder</i>, 2&3 = <i><a href="http://parts.igem.org/Part:BBa_K1618033"style="color:#002bb8;">BBa_K1618033</a></i>, 4&5 = <i><a href="http://parts.igem.org/Part:BBa_K1618029"style="color:#002bb8;">BBa_K1618029</a></i>, 6&7 = <i><a href="http://parts.igem.org/Part:BBa_K1618035"style="color:#002bb8;">BBa_K1618035</a></i>, 8&9 = <i><a href="http://parts.igem.org/Part:BBa_K1618031"style="color:#002bb8;">BBa_K1618031</a></i>, 10&11 = <i><a href="http://parts.igem.org/Part:BBa_K1618036"style="color:#002bb8;">BBa_K1618036</a></i>, 12&13 = <i><a href="http://parts.igem.org/Part:BBa_K1618032"style="color:#002bb8;">BBa_K1618032</a></i>, 14&15 = <i><a href="http://parts.igem.org/Part:BBa_K1618034"style="color:#002bb8;">BBa_K1618034</a></i>, 16&17 = <i><a href="http://parts.igem.org/Part:BBa_K1618030"style="color:#002bb8;">BBa_K1618030</a></i>, 18 = ladder. These indicate that there are no unwanted internal <i>Not</i>1 restriction sites within our constructs.</p>
 
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<div class="space30"></div>
 
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<img src="https://static.igem.org/mediawiki/2015/f/f6/NRP-UEA_Leda_2nd_Digest.jpeg" href="https://static.igem.org/mediawiki/2015/f/f6/NRP-UEA_Leda_2nd_Digest.jpeg" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 3</b>: The gel lanes are as follows – 1 = Ladder, 2&3 = <i><a href="http://parts.igem.org/Part:BBa_K1618029"style="color:#002bb8;">BBa_K1618029</a></i>, 4&5 = <i><a href="http://parts.igem.org/Part:BBa_K1618031"style="color:#002bb8;">BBa_K1618031</a></i>, 6&7 = <i><a href="http://parts.igem.org/Part:BBa_K1618032"style="color:#002bb8;">BBa_K1618032</a></i>, 8&9 = , 10&11 =<i><a href="http://parts.igem.org/Part:BBa_K1618029"style="color:#002bb8;">BBa_K1618029</a></i>, 12&13 = <i><a href="http://parts.igem.org/Part:BBa_K1618031"style="color:#002bb8;">BBa_K1618031</a></i>, 14&15 = <i><a href="http://parts.igem.org/Part:BBa_K1618032"style="color:#002bb8;">BBa_K1618032</a></i>, 16&17 = <i><a href="http://parts.igem.org/Part:BBa_K1618030"style="color:#002bb8;">BBa_K1618030</a></i>, 18 = Ladder. Lanes 2-9 were digested with Not1, while lanes 10-17 were digested with EcoR1/Pst1. The results indicate that the new tags allow our constructs to be BioBrick compatible. </p>
 
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<p class="space20">A single colony for each construct was mini-prepped and sequenced before shipping to the registry.</p>
 
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<div class="space30" id="ref"></div>
 
−
    <h2 class="title1" id="ref">References</h2>
 
−
 
−
<p >1. Patron <i>et al</i>. (2015) Standards for Plant Synthetic Biology: A Common Syntax for Exchange of DNA Parts doi: 10.1111/nph.13532/full</p>
 
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<div class="space30"></div>
 
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                        <h2 class="title1">Confirming the sub-cellular localisation of acyltransferases in plant chloroplasts</h2>
 
−
<h4 class="title2">AIM:</h4>
 
−
                        <p class="space30">To determine if our synthetic chloroplast transit peptide would direct the putative acyl trasnferases to the plant chloroplast. The aim of this experiment is to confirm that our constructs localise the acyl transferase to the plant chloroplast.  In order for the enzymes that we have chosen to acylate starch, the enzymes need to reach the chloroplast of a cell as this is where starch is produced. </p>
 
−
<h4 class="title2">METHOD:</h4>
 
−
                        <p class="space20">We transformed our binary vector constructs (see results “Making constructs to express putative acyl-transferases in plant chloroplasts” for the details of these constructs) containing the acyltransfeases with an N-terminal transit peptides and  C-terminal fluorescent reporters into <i>Agrobacterium tumefaciens</i> by electroporation (see Protocols - <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Electroporation Transformation Protocol" style = "color: #002bb8;">here</a>). Colonies were checked by PCR before liquid cultures were grown from individual colonies for infiltration of plant leaves (see Protocols - <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Agrobacterium tumefaciens" style = "color: #002bb8;">here</a>). Finally, infiltrated leaves were examined by confocal microscopy to determine the subcellular localisation of the recombinant protein.
 
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</p>
 
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                        <div class="col-md-12">
 
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<h4 class="title2">RESULTS:</h4>
 
−
                            <p class="space30">Colonies of <i>A. tumefaciens</i> were screened by PCR to confirm the presence of the binary construct (Figure 1).
 
−
</p>
 
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<img src="https://static.igem.org/mediawiki/2015/5/5e/NRP-UEA-Norwich-ledaupdated.png" href="https://static.igem.org/mediawiki/2015/5/5e/NRP-UEA-Norwich-ledaupdated.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: The gel lanes are as follows – 1 =<i><a href="http://parts.igem.org/Part:BBa_K1618029"style="color:#002bb8;">BBa_K1618029</a> control</i>, 2&3 = <i><a href="http://parts.igem.org/Part:BBa_K1618029"style="color:#002bb8;">BBa_K1618029</a> Agrobacterium</i>, 4 = <i><a href="http://parts.igem.org/Part:BBa_K1618031"style="color:#002bb8;">BBa_K1618031</a> control</i>, 5&6 = <i><a href="http://parts.igem.org/Part:BBa_K1618031"style="color:#002bb8;">BBa_K1618031</a> Agrobacterium</i>, 7 = Ladder, 8 = <i><a href="http://parts.igem.org/Part:BBa_K1618032"style="color:#002bb8;">BBa_K1618032</a> control</i>, 9&10 = <i><a href="http://parts.igem.org/Part:BBa_K1618032"style="color:#002bb8;">BBa_K1618032</a> Agrobacterium</i>, 11 = <i><a href="http://parts.igem.org/Part:BBa_K1618030"style="color:#002bb8;">BBa_K1618030</a> control</i>, 12&13 = <i><a href="http://parts.igem.org/Part:BBa_K1618030"style="color:#002bb8;">BBa_K1618030</a> Agrobacterium</i>, 14 = Ladder. The comparison between the agro-transformations and the sequence confirmed control suggests that the cloning was successful. </p>
 
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                            <p class="space30">Leaves that had been infiltrated with <i>A. tumefaciens</i> strains containing our assembled binary constructs were images using an SP5 Leica confocal microscope (Figure 2). We used two channels: the first excites chlorophyll, which is shown in red and the second excites the fluorescent fusion protein (shown in yellow).</p>
 
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<img src="https://static.igem.org/mediawiki/2015/8/89/NRP-UEA_Leda_Confocal_Microscope.png" href="https://static.igem.org/mediawiki/2015/8/89/NRP-UEA_Leda_Confocal_Microscope.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: Constructs BBa_K1618029-032 contain a yellow fluorescent protein, as well as a chloroplast transit peptide. These are confocal microscopy images of the constructs infiltrated into Nicotiana benthamiana, in which the red structures are the chlorophyll within the chloroplast, and the yellow is the fluorescent fusion protein expressed from constructs (a) <a href="http://parts.igem.org/Part:BBa_K1618029"style="color:#002bb8;">BBa_K1618029</a> (MAO enzyme), (b) <a href="http://parts.igem.org/Part:BBa_K1618031"style="color:#002bb8;">BBa_K1618031</a> (RV3034c enzyme), (c) <a href="http://parts.igem.org/Part:BBa_K1618032"style="color:#002bb8;">BBa_K1618032</a> (RV3037c enzyme), and (d) <a href="http://parts.igem.org/Part:BBa_K1618030"style="color:#002bb8;">BBa_K1618030</a> (RV3030 enzyme).
 
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</p>
 
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−
<p class="space30">Our results confirm that the putative acyltransferases  are successfully localised to the chloroplasts of the plants. </p>
 
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                                  <h2 class="title1">Determining if the expression of acyl-transferases interferes with starch accumulation in plants</h2>
 
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<h4 class="title2">AIM:</h4>
 
−
                        <p class="space30">To compare the starch content of leaves infiltrated with <i>A.tumefaciens</i> strains carrying constructs expressing our putative acyltransferases.  A potential problem of using putative acyl transferases is that they will use the same C4 position of the glucose molecules that the starch synthases add new ADP-glucose units to, interfering with growth of the starch molecule. A simple test was to confirm that normal quantities of starch were being produced. </p>
 
−
<h4 class="title2">METHOD:</h4>
 
−
                        <p class="space20">We transformed our binary vector constructs (see results “Making constructs to express putative acyl-transferases in plant chloroplasts” for the details of these constructs) containing the acyltransfeases with an N-terminal transit peptides into <i>Agrobacterium tumefaciens</i> by electroporation (see Protocols - <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Electroporation Transformation Protocol" style = "color: #002bb8;">here</a>). Colonies were checked by PCR before liquid cultures were grown from individual colonies for infiltration of plant leaves (see Protocols - <a href="https://2015.igem.org/Team:NRP-UEA-Norwich/Protocols#Agrobacterium tumefaciens" style = "color: #002bb8;">here</a>).</p>
 
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−
<p class="space20">Three plants were infiltrated with each experiment and control:</br>- <i>A. tumefaciens</i> with no construct (control)
 
−
</br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;- Strains of <i>A. tumefaciens</i> that contained each of the four binary vectors expressing putative acyltransferases.
 
−
</br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;- The plants were of the same age, same species, and were grown in the same conditions..
 
−
</p>
 
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−
<p class="space20">After infiltration, the plants were left in a normal light/dark cycle at room temperature for 24 hours before being put in the dark for 24 hours. In the dark the plants would break down the stores of starch in their leaves.</p>
 
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<p class="space20">After the dark treatment the samples from the infiltrated plants as well as control plants that had not been infiltrated were decolorized to remove the green chlorophyll and stained with iodine, which stains starch granules. A duplicate set of experiments was allowed to grow in the light for the rest of the day to build up new stores of starch. These were then assayed in the same manner to determine if expression of the putative acyltransferases interfered with starch accumulation.</p>
 
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−
<h4 class="title2">RESULTS:</h4>
 
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<p class="space20">Colonies of <i>A. tumefaciens</i> were screened by PCR to confirm the presence of the binary construct (Figure 1).</p>
 
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−
<img src="https://static.igem.org/mediawiki/2015/a/ad/NRP-UEA_Leda_agro_gel.png" href="https://static.igem.org/mediawiki/2015/a/ad/NRP-UEA_Leda_agro_gel.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: The gel lanes are as follows – 1 = Ladder, 2 = <i><a href="http://parts.igem.org/Part:BBa_K1618033"style="color:#002bb8;">BBa_K1618033</a> control</i>, 3&4 = <i><a href="http://parts.igem.org/Part:BBa_K1618033"style="color:#002bb8;">BBa_K1618033</a> Agrobacterium</i>, 5 = <i><a href="http://parts.igem.org/Part:BBa_K1618035"style="color:#002bb8;">BBa_K1618035</a> control</i>, 6&7 = <i><a href="http://parts.igem.org/Part:BBa_K1618035"style="color:#002bb8;">BBa_K1618035</a> Agrobacterium</i>, 8 = Ladder, 9 = <i><a href="http://parts.igem.org/Part:BBa_K1618036"style="color:#002bb8;">BBa_K1618036</a> control</i>, 10&11 = <i><a href="http://parts.igem.org/Part:BBa_K1618036"style="color:#002bb8;">BBa_K1618036</a> Agrobacterium</i>, 12 = <i><a href="http://parts.igem.org/Part:BBa_K1618034"style="color:#002bb8;">BBa_K1618034</a> control</i>, 13&14 = <i><a href="http://parts.igem.org/Part:BBa_K1618034"style="color:#002bb8;">BBa_K1618034</a> Agrobacterium</i>. The comparison between the agro-transformations and the sequence confirmed control suggests that the cloning was successful. </p>
 
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−
<p class="space20">After 24 hours in the dark the leaves from all samples were free of starch (<b>Figure 2A</b>). This allowed us to determine the amount of starch accumulated in the following 24 hours.  Leaves sampled after 24 hours in the light all stained dark with iodine. There is no difference in the amount of staining observed in samples from leaves infiltrated with the <i>A. tumefaciens</i> strains that contained the binary vectors expressing putative acyl transferases as compared to the control samples (<b>Figure 2B</b>).</p>
 
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<div class="space20"></div>
 
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<img src="https://static.igem.org/mediawiki/2015/0/09/NRP-UEA_Leda_Starch_content.png" href="https://static.igem.org/mediawiki/2015/0/09/NRP-UEA_Leda_Starch_content.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: Iodine staining of leaves after (A) 24 hours dark treatment, and (B) dark treatment and 8 hours light treatment. The yellow samples, indicate that no starch is present  - While there are black spots observed in some samples it is likely that these are due to damage from infiltration rather than starch. Black stain indicates the presence of starch. The level of staining doesn’t appear to differ between the different leaves and their conditions, suggesting that neither the infiltration process, or the expression of acyltransferases, affects starch production in leaves.</p>
 
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<p class="space20">These results indicate that neither the infiltration process or the expression of putative acyl-transferases has an impact on the production of starch.</p>
 
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                                  <h2 class="title1">Cloning the Butyrate Biosynthetic Pathway - PART I</h2>
 
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                        <p class="space30">Our overall aim was to clone the coding sequences (CDSs) of the butyrate biosynthetic pathway from <i>Coprococcus</i> sp. L2-50 (DSM 16842) for expression in <i>E.coli</i>. In total the pathway contains eight coding sequences. We managed to clone the pathway that would convert butyrate into butyryl-CoA (Pathway I) and, separately, the pathway that would convert acetyl-CoA into butyryl-CoA (Pathway II). </p>
 
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<img src="https://static.igem.org/mediawiki/2015/9/95/NRP-UEA-Norwich-Flavs-Thing.png" href="https://static.igem.org/mediawiki/2015/9/95/NRP-UEA-Norwich-Flavs-Thing.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<h3 class="title2">Pathway I:</h3>
 
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<h4 class="title2">AIM:</h4>
 
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                        <p class="space30">To clone the CDSs from the BUK (butyrate kinase) and PTB (<i>phosphotransbutyrylase</i>) genes from <i>Coprococcus</i> sp. with an IPTG-inducible promoter. This BioBrick is designed catalyze the conversion of butyrate from/to butyryl-CoA. The PTB gene converts butyryl-CoA from/to butyryl-phosphate and the BUK gene converts butyrate-phosphate from/to butyrate.</p>
 
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−
<div class="space20"></div>
 
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−
<h4 class="title2">METHOD:</h4>
 
−
                        <p class="space20">The CDS of the PTB and BUK coding sequences were synthesised. We digested them from their shipping plasmids with <i>XbaI</i> and <i>PstI</i>.  The pSB1C3 vector was digested with <i>EcoRI</i> and <i>PstI</i> and, the BioBrick with the LacI promoter and RBS with <i>EcoRI</i> and SpeI (See digestion protocol). The digests were run on an agarose gel and bands were gel extracted (See gel extraction protocol). The BUK CDS was ligated into the pSB1C vector together with the
 
−
IPTG-inducible promoter and RBS and the PTB CDS was added to this BioBrick to make the final pathway (<b>Figure 1</b>).
 
−
Transformation and colony screening was carried out as per our standard protocols). Putative clones were confirmed by sequencing.</p>
 
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<div class="space20"></div>
 
−
<img src="https://static.igem.org/mediawiki/2015/a/aa/NRP-UEA-Norwich-Fig1.png" href="https://static.igem.org/mediawiki/2015/a/aa/NRP-UEA-Norwich-Fig1.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: Biobrick structure of the BUJK/PTB pathway with the IPTG-inducible promoter (lacI)</p>
 
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−
<div class="space20"> </div>
 
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−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20">Successful digestion and assembly of the CDSs of the PTB and BUK genes into the PSB1C3 vector with the LacI promoter and RBS.</p>
 
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−
<p class="space20">In order to confirm that the gene inserts were successfully ligated, Colonies were screened by PCR (<b>Figure 2</b>). Putative clones were confirmed by Sanger sequencing. </p>
 
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−
<img src="https://static.igem.org/mediawiki/2015/1/19/NRP-UEA-Norwich-Fig4.png" href="https://static.igem.org/mediawiki/2015/1/19/NRP-UEA-Norwich-Fig4.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: Colony PCR products of BUK/PTB construct (lane 2-7) Primers used were BUK forward and VR. Expected size to be 1486 bp adding up to 1500 bp if considering promoter sequence.</p>
 
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                                <h2 class="title1">Cloning the Butyrate Biosynthetic Pathway - PART II</h2>
 
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                        <p class="space30">Our overall aim was to clone the coding sequences (CDSs) of the butyrate biosynthetic pathway from <i>Coprococcus</i> sp. L2-50 (DSM 16842) for expression in <i>E.coli</i>. In total the pathway contains eight coding sequences. We managed to clone the pathway that would convert butyrate into butyryl-CoA (Pathway I) and, separately, the pathway that would convert acetyl-CoA into butyryl-CoA (Pathway II). </p>
 
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<img src="https://static.igem.org/mediawiki/2015/9/95/NRP-UEA-Norwich-Flavs-Thing.png" href="https://static.igem.org/mediawiki/2015/9/95/NRP-UEA-Norwich-Flavs-Thing.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<h3 class="title2">Pathway II:</h3>
 
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<h4 class="title2">AIM:</h4>
 
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                        <p class="space30">To clone the CDSs from the <i>thiolase (THL)</i>, <i>beta-hydroxybutyryl coenzyme</i> A <i>dehydrogenase (BHBD), Crotonase (CRO), Butyryl-CoA dehydrogenase (BCD), Electron Flavoprotein subunit</i> a <i>(EFa)</i>, and <i>Electron Flavoprotein subunit</i> b <i>(EFb)</i> genes from <i>Coprococcus</i> sp. with an IPTG-inducible promoter.  This BioBrick should catalyze the conversion of acetyl-CoA to butyryl-CoA.</p>
 
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<div class="space20"> </div>
 
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<img src="https://static.igem.org/mediawiki/2015/d/d4/NRP-UEA-Norwich-Fig31.png" href="https://static.igem.org/mediawiki/2015/d/d4/NRP-UEA-Norwich-Fig31.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: Schematic of the compound part with the coding sequences for THL/BHBD/CRO/BCD/EFb/EFa (BBa_K1618021)</p>
 
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<div class="space20"> </div>
 
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−
<h4 class="title2">METHOD:</h4>
 
−
                        <p class="space20">The pSB1C3 vector was digested with <i>EcoRI</i> and <i>PstI</i> to add the <i>LacI</i> promoter and RBS, which was digested with <i>EcoRI</i> and <i>SpeI</i> (See digestion protocol). The digested plasmid was run on an agarose gel and the band was purified (See gel extraction protocol). The <i>LacI</i>-RBS part was ligated into the plasmid (See Ligation protocol) and then transformed into competent cells (See transformation protocol). The CDSs for each part of the pathway were inserted into this plasmid by digestion with <i>SpeI</i> and <i>PstI</i> and ligating in the CDSs that were released from their shipping plasmids with <i>EcoRI</i> and <i>SpeI</i>.</p>
 
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<p>Colonies were screened by PCR (see colony PCR protocol) and putative clones were miniprepped and confirmed by sequencing.</p>
 
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−
<p class="space20"> </p>
 
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−
<p class="space20"></p>
 
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−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20"><b>Successful digestion and assembly of 6 CDSs from the butyrate biosynthesis pathway into the PSB1C3 vector.</b></p>
 
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−
<p class="space20">The six genes (THL, BHBD, CRO, BCD, EFb,  EFa ) and the pSB1C3 vector with the <i>Lacl</i> promoter and RBS were digested. The expected sizes are: THL=1129 bp, BHBD=900 bp, CRO=836 bp, BCD=1211 bp, EFa=1100 bp, Efb=830 bp Figure 2 shows the successful release of parts form their backbones.</p>
 
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<img src="https://static.igem.org/mediawiki/2015/e/e1/NRP-UEA-Norwich-Fig32.png" href="https://static.igem.org/mediawiki/2015/e/e1/NRP-UEA-Norwich-Fig32.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: Agarose gel electrophoresis of restriction digestion of for the six genes of the butyrate pathway II released from their shipping plasmids as well as the backbones Lane 1=1KB Ladder, Lane 2 = pSB1C3 , Lane 3=LacI and RBS, Lane=4 PTB, Lane 5=THL, Lane 6=BCD, Lane 7=CRO, Lane 8=BHBD,Lane 9=EFa, Lane 10=EFb</p>
 
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<p class="space20">Figure 3 shows colony PCR of the THL ligated to the LacI promoter and RBS. Primers used for colony PCR and sequencing were located in the plasmid vector backbone. Expected size of THL/PRO was 1500 bp.</p>
 
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<div class="space20"></div>
 
−
<img src="https://static.igem.org/mediawiki/2015/f/f1/NRP-UEA-Norwich-Fig33.png" href="https://static.igem.org/mediawiki/2015/f/f1/NRP-UEA-Norwich-Fig33.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 3</b>: Agarose gel electrophoresis of PCR products from a colony PCR of for addition of the THL CDS to the LacI promoter and RBS. Lane 1=1KB ladder. Lane 2-8 THL/Promoter samples. Only sample 4,6 and 8 were successful.</p>
 
−
 
−
 
−
<p class="space20">Figure 4 (A) shows colony of BHBD ligated to THL, the LacI promoter and RBS .Expected size was 1100 bp. Figure 4(B) shows the digest of a successful clone. It can be observed that the band size of the digest increases each time from the previous suggesting the addition of the new insert.</p>
 
−
 
−
<div class="space20"></div>
 
−
<img src="https://static.igem.org/mediawiki/2015/6/6f/NRP-UEA-Norwich-Fig34.png" href="https://static.igem.org/mediawiki/2015/6/6f/NRP-UEA-Norwich-Fig34.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 4</b>: (A) Agarose gel electrophoresis of PCR products from a colony PCR of for addition of the BHBD CDS to the THL CDS, LacI promoter and RBS. Lane 1=1KB ladder. Lane 2-6 putative clones. (B) Agarose gel electrophoresis of digestion products of a positive clone. Lane 1=1 KB Ladder, Lane 2 Clone digest</p>
 
−
 
−
<p class="space20">Figure 5(A) shows colony of CRO ligated to BHBD, THL, the LacI promoter and RBS. Expected size was 1400 bp. Figure 5(B) shows the digest of a successful clone. It can be observed that the band size of the digest increases each time from the previous suggesting the addition of the new insert.</p>
 
−
 
−
<div class="space20"></div>
 
−
<img src="https://static.igem.org/mediawiki/2015/5/5d/NRP-UEA-Norwich-Fig35.png" href="https://static.igem.org/mediawiki/2015/5/5d/NRP-UEA-Norwich-Fig35.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 5</b>: (A) Agarose gel electrophoresis of PCR products from a colony PCR of for addition of the CRO CDS to the BHBD,THL, LacI promoter and RBS. Lane 1=1KB ladder. Lane 2-9 putative clones. Only lanes 2,3, 4, 6 and 8 were successful. (B) Agarose gel electrophoresis of digestion products of two positive clones. Lane 1=1 KB Ladder, Lanes 2-3 Digested clones </p>
 
−
 
−
<p class="space20">Figure 6(A) shows colony of BCD ligated to CRO, BHBD, THL, the LacI promoter and RBS .Expected size was 1900 bp. Figure 6(B) shows the digest of a successful clone. It can be observed that the band size of the digest increases each time from the previous suggesting the addition of the new insert.</p>
 
−
 
−
<div class="space20"></div>
 
−
<img src="https://static.igem.org/mediawiki/2015/e/e8/NRP-UEA-Norwich-Fig36.png" href="https://static.igem.org/mediawiki/2015/e/e8/NRP-UEA-Norwich-Fig36.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 6</b>: (A) Agarose gel electrophoresis of PCR products from a colony PCR of for addition of the BCD CDS to the CRO, BHBD,THL, LacI promoter and RBS. Lane 1=1KB ladder. Lane 2-3 putative clones. (B) Agarose gel electrophoresis of digestion products of two positive clones. Lane 1=1 KB Ladder, Lanes 2-3 digested clones </p>
 
−
 
−
<p class="space20">Figure 7(A) shows colony of EFb ligated to BCD, CRO, BHBD, THL, the LacI promoter and RBS. Expected size was 1500 bp. Figure 7 (B) shows the digest of a successful clone. It can be observed that the band size of the digest increases each time from the previous suggesting the addition of the new insert.</p>
 
−
 
−
<div class="space20"></div>
 
−
<img src="https://static.igem.org/mediawiki/2015/7/73/NRP-UEA-Norwich-Fig37.png" href="https://static.igem.org/mediawiki/2015/7/73/NRP-UEA-Norwich-Fig37.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 7</b>: (A) Agarose gel electrophoresis of PCR products from a colony PCR of for addition of the EFb CDS to the CRO, BCD, BHBD,THL, LacI promoter and RBS. Lane 1=1KB ladder. Lane 2-9 putative clones. Only lanes 2,3, 5, 6, 7, 8 and 9 were successful. (B) Agarose gel electrophoresis of digestion products of a positive clone. Lane 1=1 KB Ladder, Lanes 2 Digested clone.</p>
 
−
 
−
<p class="space20">Figure 8(A) shows colony of EFa ligated to EFb, BCD, CRO, BHBD, THL, the LacI promoter and RBS. Expected size was 1550 bp.</p>
 
−
 
−
<div class="space20"></div>
 
−
<img src="https://static.igem.org/mediawiki/2015/5/54/NRP-UEA-Norwich-Fig38.png" href="https://static.igem.org/mediawiki/2015/5/54/NRP-UEA-Norwich-Fig38.png" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 8</b>: (A) Agarose gel electrophoresis of PCR products from a colony PCR of for addition of the EFa CDS to the EFb, CRO, BCD, BHBD,THL, LacI promoter and RBS. Lane 1=1KB ladder. Lane 2-9 putative clones. </p>
 
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                                  <h2 class="title1">Measuring the production of butyryl coA in cultures of <i>E. coli</i></h2>
 
−
<h4 class="title2">AIM:</h4>
 
−
                        <p class="space30">To determine if BioBricks containing the two pathways (i) BUK/PTB pathway (ii) THL/BHBD/CRO/BCD/EFb/EFa pathway
 
−
could produce butyryl coA in E.coli  fed with (i) acetate or (ii) butyric acid</p>
 
−
<h4 class="title2">METHOD:</h4>
 
−
<h5 class="title2">High Pressure Liquid Chromatography (HPLC)</h5>
 
−
                        <p class="space20">In order to analyse butyryl-CoA we used HPLC. Initial development was performed using reverse phase liquid chromatography with a C18 column 100mm x 4.6mm using UV detection at 250nm and various combinations of 0.1% formic acid and acetonitrile to obtain sufficient retention.  This method was first tested on CoA and butyryl -CoA standards but no peak for CoA or butyl-CoA could be seen using this mobile phase combination. The 0.1% formic acid was replaced with water to provide a higher pH to see if this would improve the chromatography. Again no peak was seen. We suspected that the compounds may have been sensitive to active sites within the HPLC system so the water was replaced with 0.1M ammonium acetate. This resulted in a sharp peak of reasonable retention for both CoA and butyl-CoA. The final parameters used are listed below</p>
 
−
 
−
<p class="space20">Column: Luna C18 3µm 100mm x 4.6mm<br/>Detection: UV @ 250nm</p>
 
−
 
−
<p>Mobile phase:<br/>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mobile phase A: 0.1M ammonium acetate<br/>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mobile phase B: Acetonitrile</p>
 
−
 
−
<style>
 
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<body>
 
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  <tr>
 
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    <th><b>Time</b></th>
 
−
    <th><b>%A</b></th>
 
−
    <th><b>%B</b></th>
 
−
    <th><b>Flow (mL/min)</b></th>
 
−
  </tr>
 
−
  <tr>
 
−
    <td>0</td>
 
−
    <td>95</td>
 
−
    <td>5</td>
 
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    <td>1</td>
 
−
  </tr>
 
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  <tr>
 
−
    <td>95</td>
 
−
    <td>60</td>
 
−
    <td>30</td>
 
−
    <td>30</td>
 
−
  </tr>
 
−
  <tr>
 
−
    <td>5</td>
 
−
    <td>40</td>
 
−
    <td>70</td>
 
−
    <td>70</td>
 
−
  </tr>
 
−
<tr>
 
−
    <td>1</td>
 
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    <td>1</td>
 
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    <td>1</td>
 
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    <td>1</td>
 
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  </tr>
 
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<tr>
 
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    <td>1</td>
 
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    <td>1</td>
 
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    <td>1</td>
 
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    <td>1</td>
 
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  </tr>
 
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<tr>
 
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    <td>1</td>
 
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    <td>1</td>
 
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    <td>1</td>
 
−
    <td>1</td>
 
−
  </tr>
 
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</table>
 
−
 
−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20"></p>
 
−
 
−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 1</b>: </p>
 
−
 
−
<p class="space20"></p>
 
−
 
−
<div class="space20"></div>
 
−
 
−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: </p>
 
−
 
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<p class="space20"></p>
 
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<div role="tabpanel" class="tab-pane fade" id="pre4">
 
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                      <div class="col-md-12 space50">
 
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                                  <h2 class="title1">Title Last</h2>
 
−
<h4 class="title2">AIM:</h4>
 
−
                        <p class="space30"></p>
 
−
<h4 class="title2">METHOD:</h4>
 
−
                        <p class="space20"></p>
 
−
 
−
<p class="space20"> </p>
 
−
 
−
<p class="space20"></p>
 
−
 
−
<h4 class="title2">RESULTS:</h4>
 
−
<p class="space20"></p>
 
−
 
−
<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
−
<p><b>Figure 1</b>: </p>
 
−
 
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<p class="space20"></p>
 
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<div class="space20"></div>
 
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<img src="" href="" alt="..." class="img-responsive mautomargin fancybox" style="cursor: pointer;">
 
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<p><b>Figure 2</b>: </p>
 
−
 
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<p class="space20"></p>
 
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<div class="space80"></div>
 
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                    <h2 class="gray">MANY THANKS TO OUR SPONSORS</h2>
 
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Latest revision as of 15:04, 18 September 2015