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| | <div class="page-section2" id="pcontent1"> | | <div class="page-section2" id="pcontent1"> |
| − | <h2>Title</h2> | + | <h2>Cloning</h2> |
| − | <h3>Subtitle</h3>
| + | <p>The goal of the cloning experiments was to design gene constructs containing useful components for ease of cloning and protein production. LXYL-SpyTag, DBAT-SnoopTag, PAM-SnoopTag, TycA-SpyTag and mCerulean3-SnoopTag, proteins were ligated into the pET-19b expression vector via Gibson Assembly and transformed into the expression host, T7 Express E. coli. Colonies were screened for recombinant plasmids via colony PCR and sequences verified by Sanger sequencing. </p> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod | + | |
| − | tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam,
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| − | quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo
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| − | consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse
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| − | cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non
| + | |
| − | proident, sunt in culpa qui officia deserunt mollit anim id est laborum.</p>
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| | </div> | | </div> |
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| − | <div class="page-section2" id="pcontent1"> | + | <div class="page-section2" id="pcontent2"> |
| − | <h2>Title</h2> | + | <h2>Protein Expression and Purification</h2> |
| − | <h3>Subtitle</h3>
| + | <p>The goal of the protein expression and purification experiments was to express and purify the scaffolding proteins as well as the key enzymes involved in Paclitaxel production pathways. Expression was induced via IPTG induction in T7 express E. coli and proteins were purified using Immobilised Metal Affinity Chromatography (IMAC). </p> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod | + | |
| − | tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam,
| + | |
| − | quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo
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| − | consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse
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| − | cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non
| + | |
| − | proident, sunt in culpa qui officia deserunt mollit anim id est laborum.</p>
| + | |
| | </div> | | </div> |
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| − | <div class="page-section2" id="pcontent1"> | + | <div class="page-section2" id="pcontent3"> |
| − | <h2>Title</h2> | + | <h2>Scaffold Assembly</h2> |
| − | <h3>Subtitle</h3>
| + | <p>The spontaneous formation of the hexameric structure between α-prefoldin-SpyCatcher (αPFD-SpyCatcher) and β-prefoldin (βPFD-SnoopCatcher) subunits was examined by Native-PAGE (polyacrylamide gel electrophoresis). </p> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod | + | |
| − | tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam,
| + | |
| − | quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo
| + | |
| − | consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse
| + | |
| − | cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non
| + | |
| − | proident, sunt in culpa qui officia deserunt mollit anim id est laborum.</p>
| + | |
| | </div> | | </div> |
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| − | <div class="page-section2" id="pcontent1"> | + | <div class="page-section2" id="pcontent4"> |
| − | <h2>Title</h2> | + | <h2>Catcher-Tag Conjugation</h2> |
| − | <h3>Subtitle</h3>
| + | <p>To create the enzyme-scaffold complex, the alpha and beta prefoldin hexamer are to be covalently attached to the enzymes through spontaneous and specific isopeptide bond formation between the Snoop or SpyCatcher and its corresponding Snoop or SpyTag. The Catcher-Tag conjugation between βPFD-SnoopCatcher with mCerulean3 as well as αPFD-SpyCatcher with mVenus-SpyTag was examined by SDS-PAGE. </p> |
| − | <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod | + | |
| − | tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam,
| + | |
| − | quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo
| + | |
| − | consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse
| + | |
| − | cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non
| + | |
| − | proident, sunt in culpa qui officia deserunt mollit anim id est laborum.</p>
| + | |
| | </div> | | </div> |
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| | + | <div class="page-section2" id="pcontent5"> |
| | + | <h2>Enzyme Kinetics </h2> |
| | + | <p>To examine the functionality of our scaffolded system, the biosynthesis of Paclitaxel was tested. Enzyme concentration was determined by BCA assay. Ellman's assay, which is used to measure free thiol (-SH) groups in solution was used to indirectly measure of DBAT activity. |
| | + | |
| | + | The product turnover rate of PAM would have been examined by measuring the integration of the signals corresponding to alpha- and beta-phenylalanine using 1H nuclear magnetic resonance spectroscopy (1H NMR) |
| | + | |
| | + | .</p> |
| | + | </div> |
| | + | |
| | | | |
| | <!--end of section 2--> | | <!--end of section 2--> |
Experiments
Click on the parts
to see what we achieved
Scaffold
Enzymes
Click on the icons
to see how we got our results
Title
Subtitle
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Title
Subtitle
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod.
Title
Subtitle
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod.
Cloning
The goal of the cloning experiments was to design gene constructs containing useful components for ease of cloning and protein production. LXYL-SpyTag, DBAT-SnoopTag, PAM-SnoopTag, TycA-SpyTag and mCerulean3-SnoopTag, proteins were ligated into the pET-19b expression vector via Gibson Assembly and transformed into the expression host, T7 Express E. coli. Colonies were screened for recombinant plasmids via colony PCR and sequences verified by Sanger sequencing.
Protein Expression and Purification
The goal of the protein expression and purification experiments was to express and purify the scaffolding proteins as well as the key enzymes involved in Paclitaxel production pathways. Expression was induced via IPTG induction in T7 express E. coli and proteins were purified using Immobilised Metal Affinity Chromatography (IMAC).
Scaffold Assembly
The spontaneous formation of the hexameric structure between α-prefoldin-SpyCatcher (αPFD-SpyCatcher) and β-prefoldin (βPFD-SnoopCatcher) subunits was examined by Native-PAGE (polyacrylamide gel electrophoresis).
Catcher-Tag Conjugation
To create the enzyme-scaffold complex, the alpha and beta prefoldin hexamer are to be covalently attached to the enzymes through spontaneous and specific isopeptide bond formation between the Snoop or SpyCatcher and its corresponding Snoop or SpyTag. The Catcher-Tag conjugation between βPFD-SnoopCatcher with mCerulean3 as well as αPFD-SpyCatcher with mVenus-SpyTag was examined by SDS-PAGE.
Enzyme Kinetics
To examine the functionality of our scaffolded system, the biosynthesis of Paclitaxel was tested. Enzyme concentration was determined by BCA assay. Ellman's assay, which is used to measure free thiol (-SH) groups in solution was used to indirectly measure of DBAT activity.
The product turnover rate of PAM would have been examined by measuring the integration of the signals corresponding to alpha- and beta-phenylalanine using 1H nuclear magnetic resonance spectroscopy (1H NMR)
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