Difference between revisions of "Team:Macquarie Australia/Results"

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    <h1 style="text-align: center;">Project Results</h1>
 
<p>On this page we describe the results of our project and our future plans.</p>
 
<p>On this page we describe the results of our project and our future plans.</p>
  
 
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<h5>What should this page contain?</h5>
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    <h4 style="text-align: center;">What should this page contain?</h4>
 
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<li> Clearly and objectively describe the results of your work.</li>
 
<li> Clearly and objectively describe the results of your work.</li>
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<h4>Summary of achievements</h4>
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    <h4 style="text-align: center;">Summary of achievements</h4>
  
 
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<figure class="specialInline"><img src="https://static.igem.org/mediawiki/2015/9/98/MqAust_ProjectDesign_CBP.png" width="860px" alt="Chlorophyll Biosynthesis Pathway diagram"><br><br>
 
<figure class="specialInline"><img src="https://static.igem.org/mediawiki/2015/9/98/MqAust_ProjectDesign_CBP.png" width="860px" alt="Chlorophyll Biosynthesis Pathway diagram"><br><br>
<figcaption><i><b>Figure 1.</b> A schematic representation of the four designed operons containing the 13 genes necessary for the synthesis of chlorophyll-a from protoporphyrin IX in E. coli</i>.</figcaption>
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<figcaption><i>Fig 1. A schematic representation of the four designed operons containing the 13 genes necessary for the synthesis of chlorophyll-a from protoporphyrin IX in E. coli</i>.</figcaption>
 
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<p>We have successfully constructed BioBricks containing all 13 of the required genes for the synthesis of chlorophyll-a from protoporphyrin IX.
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<p align="justify">We have successfully constructed BioBricks containing all 13 of the required genes for the synthesis of chlorophyll-a from protoporphyrin IX.
 
This was achieved through the design and construction of four operons containing these 13 genes in BioBricks.
 
This was achieved through the design and construction of four operons containing these 13 genes in BioBricks.
 
The four operons were constructed by assembling our 13 BioBricks into composite parts using a <a href="https://2015.igem.org/Team:Macquarie_Australia/Experiments">restriction digestion and ligation protocol</a>.
 
The four operons were constructed by assembling our 13 BioBricks into composite parts using a <a href="https://2015.igem.org/Team:Macquarie_Australia/Experiments">restriction digestion and ligation protocol</a>.
 
All of our assembled parts were sequenced for further confirmation of our success.</p>
 
All of our assembled parts were sequenced for further confirmation of our success.</p>
 
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<img src="https://static.igem.org/mediawiki/2015/9/90/Image_1_results_1_gm_mq.jpg">
 
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<figcaption><i><b>Figure 2.</b> Single (EcoRI) and double (EcoRI + PstI) restriction digests of four operon constructs containing 13 genes required for the chlorophyll-a synthesis pathway on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.</i></figcaption>
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<figcaption><i>Fig 2. Single (EcoRI) and double (EcoRI + PstI) restriction digests of four operon constructs containing 13 genes required for the chlorophyll-a synthesis pathway on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.</i></figcaption>
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<h5>ChlH</h5>
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    <h4 style="text-align: center;">ChlH</h4>
<p>We successfully constructed the ChlH(BBa_K1080001)(4166kb) BioBrick. ChlH is part of operon 1 constituting the chlorophyll-a biosynthesis pathway in <i>E. coli</i>.</p>
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<p align="justify">We successfully constructed the ChlH(BBa_K1080001)(4166kb) BioBrick. ChlH is part of operon 1 constituting the chlorophyll-a biosynthesis pathway in <i>E. coli</i>.</p>
<p>The ChlH construct’s purpose is to generate Mg-protoporphyrin IX from Protoporphyrin IX, as the very first step towards synthesising chlorophyll-a from Protoporphyrin IX<sup>(1)</sup>. The role of ChlH is to chelate Mg<sup>2+</sup> to Protoporphyrin IX, thereby synthesising MG-protoporphyrin IX <sup>(1)</sup>.</p>
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<p align="justify">The ChlH construct’s purpose is to generate Mg-protoporphyrin IX from Protoporphyrin IX, as the very first step towards synthesising chlorophyll-a from Protoporphyrin IX<sup>(1)</sup>. The role of ChlH is to chelate Mg<sup>2+</sup> to Protoporphyrin IX, thereby synthesising MG-protoporphyrin IX <sup>(1)</sup>.</p>
<p>ChlH was synthesized in three fragments (figure 3), and assembled in two steps First G13, 3-6 and the KAN vector were combined via restriction digest and then P2 was added via Gibson assembly(2) (figure 4).</p>
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<p align="justify">ChlH was synthesized in three fragments (figure 3), and assembled in two steps First G13, 3-6 and the KAN vector were combined via restriction digest and then P2 was added via Gibson assembly(2) (figure 4).</p>
  
 
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<figcaption><i><b>Figure 3.</b> Restriction digest of gibson assembly product visualised on a 1% agarose gel stained with GelRed. Lane 1 is a 1 kb a standard molecular ladder. Lane 2 is a single digest by EcoRI and Lane 3 is a double digest by EcoRI and PstI. A band of approximately 6 kb in lane 1 corresponds with the expected size for the linearized KAN vector and assembled ChlH fragments. Bands at approximately 2 kb and 4 kb correspond with the expected size of the KAN vector and ChlH gene respectively.</i></figcaption>
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<figcaption><i>Fig 3. Restriction digest of gibson assembly product visualised on a 1% agarose gel stained with GelRed. Lane 1 is a 1 kb a standard molecular ladder. Lane 2 is a single digest by EcoRI and Lane 3 is a double digest by EcoRI and PstI. A band of approximately 6 kb in lane 1 corresponds with the expected size for the linearized KAN vector and assembled ChlH fragments. Bands at approximately 2 kb and 4 kb correspond with the expected size of the KAN vector and ChlH gene respectively.</i></figcaption>
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<h5>Protein expression</h5>
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    <h4 style="text-align: center;">Protein expression</h4><br>
<p>We have successful expressed the ChlM and GUN4 genes in <i>E. coli</i>. This was confirmed by running protein extracts on an SDS-PAGE gel, which showed distinct gel bands corresponding to the theoretical sizes of these three proteins (figure 4).<p>
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<p align="justify">We have successful expressed the ChlM and GUN4 genes in <i>E. coli</i>. This was confirmed by running protein extracts on an SDS-PAGE gel, which showed distinct gel bands corresponding to the theoretical sizes of these three proteins (figure 4).<p>
  
 
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<figcaption><i><b>Figure 4.</b> SDS-PAGE protein expression analysis of transformed <i>E. coli</i> cells each expressing ChlM and GUN4. Protein extracts were loaded onto 4-12% Bis-Tris gel. A unique band is present at the molecular weight of approximately 30kDa (theoretical mass of ChlM) signifying expression of S-Adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase. This was further confirmed by enzymatic functional assay. Overexpression of GUN4 was also observed (as highlighted), and functionality proven by a simple fluorescence spectrophotometric measurement. </i></figcaption>
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<figcaption><i>Fig 4. SDS-PAGE protein expression analysis of transformed <i>E. coli</i> cells each expressing ChlM and GUN4. Protein extracts were loaded onto 4-12% Bis-Tris gel. A unique band is present at the molecular weight of approximately 30kDa (theoretical mass of ChlM) signifying expression of S-Adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase. This was further confirmed by enzymatic functional assay. Overexpression of GUN4 was also observed (as highlighted), and functionality proven by a simple fluorescence spectrophotometric measurement. </i></figcaption>
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<h5>Functional assay of submitted parts</h5>
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    <h4 style="text-align: center;">Functional assay of submitted parts</h4>
<p>We have demonstrated the functionality of ChlM enzyme encoding magnesium protoporphyrin IX methyltransferase (lac+ChlM: BBa_K1640018), an enzyme within the third operon in the chlorophyll-a biosynthesis pathway.  ChlM catalyses the methylation of a carboxyl group in magnesium-protoporphyrin IX using cofactor S-Adenosyl-L-methionine (SAM), yielding magnesium-protophorphyrin IX mono-methyl ester (3);</p>
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<p align="justify">We have demonstrated the functionality of ChlM enzyme encoding S-Adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase (SAM:MgPMT) (lac+ChlM: BBa_K1640018), an enzyme within the third operon in the chlorophyll-a biosynthesis pathway.  SAM catalyses the methylation of a carboxyl group in magnesium-protoporphyrin IX, yielding magnesium-protophorphyrin IX mono-methyl ester(3);</p>
<p>S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.</p>
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<p align="justify">S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.</p>
  
 
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<figcaption><i><b>Figure 5.</b> Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).</i></figcaption>
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<figcaption><i>Fig 5. Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).</i></figcaption>
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<p align="justify">To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into E. coli strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).</p>
  
<p>To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into E. coli strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).</p>
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    <h4 style="text-align: center;">Functional assay of ChlM</h4>
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<h5>Functional assay of ChlM</h5>
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<p>Cell lysate containing over expressed ChlM was added with substrates and products were separated by FPLC (Figure 3).</p>
 
<p>Cell lysate containing over expressed ChlM was added with substrates and products were separated by FPLC (Figure 3).</p>
  
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<figcaption><i><b>Figure 6.</b> FPLC chromatogram of the products of ChlM activity alongside positive and negative controls. The substrate MG-PPIX elutes at 3.04 mins, while the product Mg-PPIX mono-ester elutes at 4.22 mins.</i></figcaption>
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<figcaption><i>Fig 6. FPLC chromatogram of the products of ChlM activity alongside positive and negative controls. The substrate MG-PPIX elutes at 3.04 mins, while the product Mg-PPIX mono-ester elutes at 4.22 mins.</i></figcaption>
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<p align="justify">The FPLC chromatogram data enabled determination of ChlM enzyme activity (Figure 3). This data enabled further determination of levels of Mg-Protoporphyrin IX converted to Mg-Protoporphyrin IX-ME by ChlM. In this instance, 26% of the precursor molecule Mg-Protoporphyrin IX had been catalysed by the enzyme ChlM into Mg-Protoporphyrin IX-ME.</p>
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<p align="justify">When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.</p>
  
<p>The FPLC chromatogram data enabled determination of ChlM enzyme activity (Figure 3). This data enabled further determination of levels of Mg-Protoporphyrin IX converted to Mg-Protoporphyrin IX-ME by ChlM. In this instance, 26% of the precursor molecule Mg-Protoporphyrin IX had been catalysed by the enzyme ChlM into Mg-Protoporphyrin IX-ME.</p>
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<h4 style="text-align: center;">Functional Assay of GUN4</h4>
<p>When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.</p>
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<p align="justify"> The gene GUN4 encodes a cofactor that binds to protoporphyrin IX facilitating the Mg-chelatase mediated insertion of an Mg2+ ion to form Mg-protoporphyrin IX. Protoporphyrin IX has fluorescent properties with an excitation spectrum that peaks at 404nm and an emission peak at 635nm.</p>
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<p align="justify">The assay undertaken involved adding protoporphyrin IX to cell lysates overexpressing GUN4 and exposing these samples to light at a wavelength of 280nm. This excites tryptophan residues in GUN4 and, when bound, the the resulting 340nm fluorescence will excite protoporphyrin IX, resulting in an emission peak at 635nm that can be detected by a spectrophotometer.</p>
<h5>Functional Assay of GUN4</h5>
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<p align="justify">For this assay, cells were lysed through use of a French press and these lysates were diluted ten-fold to approximately 0.2mg/ml concentration of protein. Protoporphyrin was prepared to a concentration of 2𝛍M and added to samples of lysate containing 0µg, 5µg, 10µg, 15µg and 20µg of GUN4. A non-specific protein control (Bovine Serum Albumin) and heat treated GUN4 cell lysates (80 degrees Celsius for ten minutes) were used as controls.</p>
        <p> The gene GUN4 encodes a cofactor that binds to protoporphyrin IX facilitating the Mg-chelatase mediated insertion of an Mg2+ ion to form Mg-protoporphyrin IX. Protoporphyrin IX has fluorescent properties with a wide excitation spectrum that peaks at 404nm and an emission peak at 635nm.</p>
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<p align="justify">When excited at 280nm, emission peaks at 635nm were detected through fluorescence spectrophotometry for each GUN4 lysate, with increased intensity as GUN4 concentration rose. BSA showed no fluorescent activity when excited at this wavelength and the heat treated lysates displayed a much lower level of fluorescence compared to the unheated samples (Figure 11.).</p>
        <p>The assay undertaken involved adding protoporphyrin IX to cell lysates overexpressing GUN4 and exposing these samples to light at a wavelength of 280nm. This excites tryptophan residues in GUN4 and, when bound, the the resulting 340nm fluorescence will excite protoporphyrin IX, resulting in an emission peak at 635nm that can be detected by a spectrophotometer.</p>
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<img src="https://static.igem.org/mediawiki/2015/3/3d/PPIX_peaks.jpg/669px-PPIX_peaks.jpg">
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<figcaption><i><b>Figure x.</b>The emission spectra of GUN over-expressing lysates, heat treated lysates and BSA (negative control) when excited at 280nm. Emission wavelengths from 580nm to 800nm were measured, and the emission maxima for protein bound PPIX is 635nm.</i></figcaption>
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<figcaption><i>Fig 8. Plots of fluorescence intensity at emission 635nm against protein concentration. Gun4 showed a clear concentration dependent relationship with PPIX binding, which was reduced after a short heat treatment. BSA acts as a negative control</i></figcaption>
 
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<br>
        <p>For this assay, cells were lysed through use of a French press and these lysates were diluted ten-fold to approximately 0.2mg/ml concentration of protein. Protoporphyrin was prepared to a concentration of 2𝛍M and added to samples of lysate containing 0µg, 5µg, 10µg, 15µg and 20µg of GUN4. A non-specific protein control (Bovine Serum Albumin) and heat treated GUN4 cell lysates (80 degrees Celsius for ten minutes) were used as controls.</p>
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<p align="justify">This demonstrates that GUN4 is biologically active, binding to protoporphyrin IX. Further, the linear increase in emission at 635nm with greater concentration of GUN4 is indicative of higher rates of binding. This corresponds with the modelling conducted by the Macquarie University team of 2014 that suggests a linear pattern of protoporphyrin IX/GUN4 complex formation. Our functional assay builds upon this framework as we found that this linear relationship only occurs at low protein concentrations, when the lysates were diluted to a 0.2mg/ml.</p>  
        <p>When excited at 280nm, emission peaks at 635nm were detected through fluorescence spectrophotometry for each GUN4 lysate, with increased intensity as GUN4 concentration rose. BSA showed no fluorescent activity when excited at this wavelength and the heat treated lysates displayed a much lower level of fluorescence compared to the unheated samples (Figure 11.).</p>
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    <h4 style="text-align: center;">Mass Spectrometry Analysis of ChlM</h4>
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<figcaption><i><b>Figure 11.</b> Plots of fluorescence intensity at emission 635nm against protein concentration. Gun4 showed a clear concentration dependent relationship with PPIX binding, which was reduced after a short heat treatment. BSA acts as a negative control</i></figcaption>
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        <p>This demonstrates that GUN4 is biologically active, binding to protoporphyrin IX. Further, the linear increase in emission at 635nm with greater concentration of GUN4 is indicative of higher rates of binding. This corresponds with the modelling conducted by the Macquarie University team of 2014 that suggests a linear pattern of protoporphyrin IX/GUN4 complex formation. Our functional assay builds upon this framework as we found that this linear relationship only occurs at low protein concentrations, when the lysates were diluted to a 0.2mg/ml.</p>  
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<h5>Mass Spectrometry Analysis of ChlM</h5>
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<p>Upon expression of ChlM (see Figure 4) mass spectrometry analysis of excised and trypsin digested gel band at 30kDa with ESI found peptides matching.</p>
 
<p>Upon expression of ChlM (see Figure 4) mass spectrometry analysis of excised and trypsin digested gel band at 30kDa with ESI found peptides matching.</p>
  
<h5>Photosystem II</h5>
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    <h4 style="text-align: center;">Photosystem II</h4>
<p>We designed 5 operons consisting of a total of 17 genes from <i>C. reinhardtii</i> which encoded for essential proteins for Photosystem II (see table 1). We divided these 17 genes arbitrarily into 10 BioBricks.</p><br><br>
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<p align="justify">We designed 5 operons consisting of a total of 17 genes from <i>C. reinhardtii</i> which encoded for essential proteins for Photosystem II (see table 1). We divided these 17 genes arbitrarily into 10 BioBricks.</p><br><br>
  
 
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<img src="https://static.igem.org/mediawiki/2015/1/14/MqAust_ProjectDesign_PSB.png" width="860px" alt="Photosystem II diagram">
 
<img src="https://static.igem.org/mediawiki/2015/1/14/MqAust_ProjectDesign_PSB.png" width="860px" alt="Photosystem II diagram">
 
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<figcaption><i><b>Figure 7.</b> A schematic representation of 5 designed operons containing the 17 necessary genes for biosynthesis of Photosystem II in E. coli.</i></figcaption>
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<figcaption><i>Fig 9. A schematic representation of 5 designed operons containing the 17 necessary genes for biosynthesis of Photosystem II in E. coli.</i></figcaption>
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<p align="justify">Our team successfully constructed 8 of the 10 BioBricks designed for Photosystem II. These were confirmed by sequencing.</p>
  
<p>Our team successfully constructed 8 of the 10 BioBricks designed for Photosystem II. These were confirmed by sequencing.</p>
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<p align="justify">Table 2. The 10 BioBricks we attempted to create, whether we were able to detect a constituent gene fragment in a gel-electrophoresis and whether confirmed an accurate sequence from an extracted plasmid.</p>
  
<p>Table 2. The 10 BioBricks we attempted to create, whether we were able to detect a constituent gene fragment in a gel-electrophoresis and whether confirmed an accurate sequence from an extracted plasmid.</p>
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<table class="regularTable" style="width:40%" cellpadding="0" align="center">
 
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<th class="regularCol20" style="text-align:center">Biobrick</th>
 
<th class="regularCol20" style="text-align:center">Biobrick</th>
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<figcaption><i><b>Figure 8.</b> Single (EcoRI) and double (EcoRI + PstI) restriction digests of eight BioBricks containing 14 genes required for the construction of PSII on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.</i></figcaption>
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<figcaption><i>Fig 10. Single (EcoRI) and double (EcoRI + PstI) restriction digests of eight BioBricks containing 14 genes required for the construction of PSII on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.</i></figcaption>
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<p align="justify"> If we achieve this we can then transform <i>E. coli</i> which we have engineered to develop chlorophyll and hydrogenase, this will allow the <i>E. coli</i> to produce a functioning PSII and allow the production of H<sub>2</sub>.<p>
  
<p>If we achieve this we can then transform <i>E. coli</i> which we have engineered to develop chlorophyll and hydrogenase, this will allow the <i>E. coli</i> to produce a functioning PSII and allow the production of H<sub>2</sub>.<p>
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<h4 style="text-align: center;">Modelling</h4>
 
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<p align="justify"> ALA is the first precursor on the pathway to biosynthesis of both chlorophyll-a and heme (6). As an organism which produces its own heme, E. coli itself produces 5-ALA and converts it to PPIX (the point of divergence between the heme and chlorophyll-a synthesis pathways) through a multi-step pathway for which it naturally possesses the necessary genes (6).</p>
<h5>Modelling</h5>
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<p align="justify"> We successfully modelled the production of PPIX from 5-ALA in non-genetically transformed E. coli, comparing how different concentrations of the precursor 5-ALA affected the production of PPIX (figure 9).</p>
<p>ALA is the first precursor on the pathway to biosynthesis of both chlorophyll-a and heme (6). As an organism which produces its own heme, E. coli itself produces 5-ALA and converts it to PPIX (the point of divergence between the heme and chlorophyll-a synthesis pathways) through a multi-step pathway for which it naturally possesses the necessary genes (6).</p>
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<p>We successfully modelled the production of PPIX from 5-ALA in non-genetically transformed E. coli, comparing how different concentrations of the precursor 5-ALA affected the production of PPIX (figure 9).</p>
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<figcaption><i><b>Figure 9.</b> Concentration of PPIX produced from various initial concentrations of 5-ALA for E. coli cell culture concentrations of 0.2mM, with MgCl added at a concentration of 2mM.</i></figcaption><br><br>
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<figcaption><i>Fig 11. Concentration of PPIX produced from various initial concentrations of 5-ALA for E. coli cell culture concentrations of 0.2mM, with MgCl added at a concentration of 2mM.</i></figcaption><br><br>
  
 
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<figcaption><i><b>Figure 10.</b> A schematic representation of Protoporphyrinogen IX causing substrate inhibition of 5-ALA’S conversion to porphobilinogen via porphobilinogen synthase.</i></figcaption>
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<figcaption><i>Fig 12. A schematic representation of Protoporphyrinogen IX causing substrate inhibition of 5-ALA’S conversion to porphobilinogen via porphobilinogen synthase.</i></figcaption>
 
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<h5>Future Direction of our Project</h5>
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    <h4 style="text-align: center;">Future Direction of our Project</h4>
 
<ul>
 
<ul>
 
<li>To demonstrate functionality of our constructed parts for chlorophyll-a synthesis, through successfully inducing protein expression of, and functionally characterising, all 13 genes in the pathway.</li>
 
<li>To demonstrate functionality of our constructed parts for chlorophyll-a synthesis, through successfully inducing protein expression of, and functionally characterising, all 13 genes in the pathway.</li>
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<li>To successfully biosynthesise a hydrogenase enzyme, allowing for the combination of electrons and protons for the synthesis of hydrogen gas. This would help us build a functional prototype.</li>
 
<li>To successfully biosynthesise a hydrogenase enzyme, allowing for the combination of electrons and protons for the synthesis of hydrogen gas. This would help us build a functional prototype.</li>
 
</ul>  
 
</ul>  
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<h5>References</h5>
 
<h5>References</h5>
 
<ol>
 
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Revision as of 02:21, 19 September 2015

Results
Link to Project Description page
Link to Experiments & Protocols page
Results page
Link to Notebook page
Link to Safety page

Project Results

On this page we describe the results of our project and our future plans.


Summary of achievements

  • Successful completion of the chlorophyll-a synthesis pathway - all 13 genes required for chlorophyll-a synthesis in E. coli successfully placed into BioBricks
  • Functional characterisation of the ChlM enzyme. Functionality was demonstrated in converting Magnesium-Protoporphyrin IX (Mg-PPIX) to Magnesium-Protoporphyrin IX-Monomethyl Ester (Mg-PPIX-ME). The presence of ChlM further confirmed by running of extracted protein on an SDS-PAGE gel
  • Improved the characterization of Gun4 (BBa_K1080003) by showing PPIX binding with an alternate, simpler method and documented it as a contribution under the experience page
  • Successfully constructed BioBricks containing 14 of the 17 genes required for biosynthesis of Photosystem II
  • Modelling of pathway from 5-aminolevulinic acid to PPIX. This determined what concentration of 5-aminolevulinic acid (5-ALA) resulted in optimum yield of Protoporphyrin IX (PPIX)


Chlorophyll Biosynthesis Pathway diagram

Fig 1. A schematic representation of the four designed operons containing the 13 genes necessary for the synthesis of chlorophyll-a from protoporphyrin IX in E. coli.

We have successfully constructed BioBricks containing all 13 of the required genes for the synthesis of chlorophyll-a from protoporphyrin IX. This was achieved through the design and construction of four operons containing these 13 genes in BioBricks. The four operons were constructed by assembling our 13 BioBricks into composite parts using a restriction digestion and ligation protocol. All of our assembled parts were sequenced for further confirmation of our success.



Fig 2. Single (EcoRI) and double (EcoRI + PstI) restriction digests of four operon constructs containing 13 genes required for the chlorophyll-a synthesis pathway on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.

ChlH

We successfully constructed the ChlH(BBa_K1080001)(4166kb) BioBrick. ChlH is part of operon 1 constituting the chlorophyll-a biosynthesis pathway in E. coli.

The ChlH construct’s purpose is to generate Mg-protoporphyrin IX from Protoporphyrin IX, as the very first step towards synthesising chlorophyll-a from Protoporphyrin IX(1). The role of ChlH is to chelate Mg2+ to Protoporphyrin IX, thereby synthesising MG-protoporphyrin IX (1).

ChlH was synthesized in three fragments (figure 3), and assembled in two steps First G13, 3-6 and the KAN vector were combined via restriction digest and then P2 was added via Gibson assembly(2) (figure 4).



Fig 3. Restriction digest of gibson assembly product visualised on a 1% agarose gel stained with GelRed. Lane 1 is a 1 kb a standard molecular ladder. Lane 2 is a single digest by EcoRI and Lane 3 is a double digest by EcoRI and PstI. A band of approximately 6 kb in lane 1 corresponds with the expected size for the linearized KAN vector and assembled ChlH fragments. Bands at approximately 2 kb and 4 kb correspond with the expected size of the KAN vector and ChlH gene respectively.

Protein expression


We have successful expressed the ChlM and GUN4 genes in E. coli. This was confirmed by running protein extracts on an SDS-PAGE gel, which showed distinct gel bands corresponding to the theoretical sizes of these three proteins (figure 4).


Fig 4. SDS-PAGE protein expression analysis of transformed E. coli cells each expressing ChlM and GUN4. Protein extracts were loaded onto 4-12% Bis-Tris gel. A unique band is present at the molecular weight of approximately 30kDa (theoretical mass of ChlM) signifying expression of S-Adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase. This was further confirmed by enzymatic functional assay. Overexpression of GUN4 was also observed (as highlighted), and functionality proven by a simple fluorescence spectrophotometric measurement.

Functional assay of submitted parts

We have demonstrated the functionality of ChlM enzyme encoding S-Adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase (SAM:MgPMT) (lac+ChlM: BBa_K1640018), an enzyme within the third operon in the chlorophyll-a biosynthesis pathway. SAM catalyses the methylation of a carboxyl group in magnesium-protoporphyrin IX, yielding magnesium-protophorphyrin IX mono-methyl ester(3);

S-adenosyl-L-methionine + magnesium protoporphyrin IX ⇌ S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.


Fig 5. Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).

To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into E. coli strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).

Functional assay of ChlM

Cell lysate containing over expressed ChlM was added with substrates and products were separated by FPLC (Figure 3).


Fig 6. FPLC chromatogram of the products of ChlM activity alongside positive and negative controls. The substrate MG-PPIX elutes at 3.04 mins, while the product Mg-PPIX mono-ester elutes at 4.22 mins.

The FPLC chromatogram data enabled determination of ChlM enzyme activity (Figure 3). This data enabled further determination of levels of Mg-Protoporphyrin IX converted to Mg-Protoporphyrin IX-ME by ChlM. In this instance, 26% of the precursor molecule Mg-Protoporphyrin IX had been catalysed by the enzyme ChlM into Mg-Protoporphyrin IX-ME.

When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.

Functional Assay of GUN4

The gene GUN4 encodes a cofactor that binds to protoporphyrin IX facilitating the Mg-chelatase mediated insertion of an Mg2+ ion to form Mg-protoporphyrin IX. Protoporphyrin IX has fluorescent properties with an excitation spectrum that peaks at 404nm and an emission peak at 635nm.

The assay undertaken involved adding protoporphyrin IX to cell lysates overexpressing GUN4 and exposing these samples to light at a wavelength of 280nm. This excites tryptophan residues in GUN4 and, when bound, the the resulting 340nm fluorescence will excite protoporphyrin IX, resulting in an emission peak at 635nm that can be detected by a spectrophotometer.

For this assay, cells were lysed through use of a French press and these lysates were diluted ten-fold to approximately 0.2mg/ml concentration of protein. Protoporphyrin was prepared to a concentration of 2𝛍M and added to samples of lysate containing 0µg, 5µg, 10µg, 15µg and 20µg of GUN4. A non-specific protein control (Bovine Serum Albumin) and heat treated GUN4 cell lysates (80 degrees Celsius for ten minutes) were used as controls.

When excited at 280nm, emission peaks at 635nm were detected through fluorescence spectrophotometry for each GUN4 lysate, with increased intensity as GUN4 concentration rose. BSA showed no fluorescent activity when excited at this wavelength and the heat treated lysates displayed a much lower level of fluorescence compared to the unheated samples (Figure 11.).


Fig 8. Plots of fluorescence intensity at emission 635nm against protein concentration. Gun4 showed a clear concentration dependent relationship with PPIX binding, which was reduced after a short heat treatment. BSA acts as a negative control

This demonstrates that GUN4 is biologically active, binding to protoporphyrin IX. Further, the linear increase in emission at 635nm with greater concentration of GUN4 is indicative of higher rates of binding. This corresponds with the modelling conducted by the Macquarie University team of 2014 that suggests a linear pattern of protoporphyrin IX/GUN4 complex formation. Our functional assay builds upon this framework as we found that this linear relationship only occurs at low protein concentrations, when the lysates were diluted to a 0.2mg/ml.

Mass Spectrometry Analysis of ChlM

Upon expression of ChlM (see Figure 4) mass spectrometry analysis of excised and trypsin digested gel band at 30kDa with ESI found peptides matching.

Photosystem II

We designed 5 operons consisting of a total of 17 genes from C. reinhardtii which encoded for essential proteins for Photosystem II (see table 1). We divided these 17 genes arbitrarily into 10 BioBricks.



Photosystem II diagram


Fig 9. A schematic representation of 5 designed operons containing the 17 necessary genes for biosynthesis of Photosystem II in E. coli.

Our team successfully constructed 8 of the 10 BioBricks designed for Photosystem II. These were confirmed by sequencing.

Table 2. The 10 BioBricks we attempted to create, whether we were able to detect a constituent gene fragment in a gel-electrophoresis and whether confirmed an accurate sequence from an extracted plasmid.

Biobrick Operon Constructed
PsbE-L-J 2 Y
PsbQ-R 5 Y
PsbM-Z-H 4 Y
PsbA 1 Y
PsbW-K 4 Y
PsbP 5 Y
PsbO 5 Y
PsbC 1 Y
PsbT-B 3 N
PsbD 1 N


Fig 10. Single (EcoRI) and double (EcoRI + PstI) restriction digests of eight BioBricks containing 14 genes required for the construction of PSII on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.

If we achieve this we can then transform E. coli which we have engineered to develop chlorophyll and hydrogenase, this will allow the E. coli to produce a functioning PSII and allow the production of H2.

Modelling

ALA is the first precursor on the pathway to biosynthesis of both chlorophyll-a and heme (6). As an organism which produces its own heme, E. coli itself produces 5-ALA and converts it to PPIX (the point of divergence between the heme and chlorophyll-a synthesis pathways) through a multi-step pathway for which it naturally possesses the necessary genes (6).

We successfully modelled the production of PPIX from 5-ALA in non-genetically transformed E. coli, comparing how different concentrations of the precursor 5-ALA affected the production of PPIX (figure 9).


Fig 11. Concentration of PPIX produced from various initial concentrations of 5-ALA for E. coli cell culture concentrations of 0.2mM, with MgCl added at a concentration of 2mM.



Fig 12. A schematic representation of Protoporphyrinogen IX causing substrate inhibition of 5-ALA’S conversion to porphobilinogen via porphobilinogen synthase.

Future Direction of our Project

  • To demonstrate functionality of our constructed parts for chlorophyll-a synthesis, through successfully inducing protein expression of, and functionally characterising, all 13 genes in the pathway.
  • To finish the construction of Photosystem II. This would be through the construction of BioBricks for the remaining components (PsbD and PsbT-B), building of composite parts by joining the constructed BioBricks, and the development of a BioBrick containing all 5 operons.
  • To successfully biosynthesise a hydrogenase enzyme, allowing for the combination of electrons and protons for the synthesis of hydrogen gas. This would help us build a functional prototype.


References
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