Difference between revisions of "Team:UNSW Australia/Experiments"

Line 114: Line 114:
  
 
<div class="page-section2" id="pcontent1">
 
<div class="page-section2" id="pcontent1">
−
<h2>Cloning</h2>
+
<h2>Laboratory Objective #1 - Produce the Assemblase Scaffold</h2>
−
<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>This is the first of our three laboratory objectives. We aim to express and purify βPFD-SnoopCatcher and αPFD-SpyCatcher. The assembly of these proteins into the Assemblase complex will then be demonstrated. Conjugation of SpyTag to SpyCatcher and SnoopTag to SnoopCatcher will also be demonstrated.  
 +
To learn more see our Assemblase <a href="https://2019.igem.org/Team:UNSW_Australia/Scaffold">Assemblase Scaffold</a> page.
 +
</p>
 
</div>  
 
</div>  
  
  
 
<div class="page-section2" id="pcontent2">
 
<div class="page-section2" id="pcontent2">
−
<h2>Protein Expression and Purification</h2>
+
<h2>Laboratory Objective #2 - Produce and test PAM and TycA of Pathway 1</h2>
−
<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>Our second lab objective was to assemble and test pathway 1. We aimed to express and purify the reaction enzymes involved in the biosynthesis of the Paclitaxel side-chain; PAM-SnoopTag and TycA-SpyTag. These enzymes will then be conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher, respectively. The catalytic activity of the enzymes will also be tested. To learn more see our <a href="https://2019.igem.org/Team:UNSW_Australia/Pathway1">Pathway 1</a> page. </p>
 
</div>  
 
</div>  
  
 
<div class="page-section2" id="pcontent3">
 
<div class="page-section2" id="pcontent3">
−
<h2>Scaffold Assembly</h2>
+
<h2>Laboratory Objective #3 - Produce and test LXYL and DBAT of Pathway 2</h2>
−
<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>This is the third of our three laboratory objectives. We aim to express and purify the reaction enzymes involved in the alternative biosynthesis of Paclitaxel; DBAT-SnoopTag and LXYL-SpyTag. These enzymes will then be conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher, respectively. The catalytic activity of the enzymes will also be tested. To learn more see our <a href="https://2019.igem.org/Team:UNSW_Australia/Pathway2">Pathway 2</a> page. </p>
−
</div>  
+
  
  
−
<div class="page-section2" id="pcontent4">
 
−
<h2>Catcher-Tag Conjugation</h2>
 
−
<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>
 
 
</div>  
 
</div>  
  
  
  
−
<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>
 
.</p>

Revision as of 14:33, 21 October 2019


Team: UNSW Australia


Experiments

scaffoldnotingrey11

Click on the icons

to see how we got our results

map1

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.

Title

Subtitle

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod.

Laboratory Objective #1 - Produce the Assemblase Scaffold

This is the first of our three laboratory objectives. We aim to express and purify βPFD-SnoopCatcher and αPFD-SpyCatcher. The assembly of these proteins into the Assemblase complex will then be demonstrated. Conjugation of SpyTag to SpyCatcher and SnoopTag to SnoopCatcher will also be demonstrated. To learn more see our Assemblase Assemblase Scaffold page.

Laboratory Objective #2 - Produce and test PAM and TycA of Pathway 1

Our second lab objective was to assemble and test pathway 1. We aimed to express and purify the reaction enzymes involved in the biosynthesis of the Paclitaxel side-chain; PAM-SnoopTag and TycA-SpyTag. These enzymes will then be conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher, respectively. The catalytic activity of the enzymes will also be tested. To learn more see our Pathway 1 page.

Laboratory Objective #3 - Produce and test LXYL and DBAT of Pathway 2

This is the third of our three laboratory objectives. We aim to express and purify the reaction enzymes involved in the alternative biosynthesis of Paclitaxel; DBAT-SnoopTag and LXYL-SpyTag. These enzymes will then be conjugated to βPFD-SnoopCatcher and αPFD-SpyCatcher, respectively. The catalytic activity of the enzymes will also be tested. To learn more see our Pathway 2 page.

.