Difference between revisions of "Team:Cardiff Wales/Collaborations"

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<h2>FLIM experiments with Oxford iGEM</h2>
 
<h2>FLIM experiments with Oxford iGEM</h2>
 
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<p>The Oxford iGEM team wanted to test their copper chelators using Fluorescence Lifetime IMaging (FLIM), previous research by <a href= https://www.ncbi.nlm.nih.gov/pubmed/21773693/"target=_blank"><I>Hötzer et al</I></a> (2011) had shown that GFP lifetime was modified in a copper concentration dependent manner. The <a href="http://blogs.cardiff.ac.uk/bioimaging/flim/" target="_blank">Cardiff Bioimaging</a> facility has a microscope that can perform these reactions so we offered to test their samples.</p>
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<p>The Oxford iGEM team wanted to test their copper chelators using Fluorescence Lifetime IMaging (FLIM), previous research by <a href= https://www.ncbi.nlm.nih.gov/pubmed/21773693/"target=_blank">Hötzer <I>et al</I> (2011)</a>had shown that GFP lifetime was modified in a copper concentration dependent manner. The <a href="http://blogs.cardiff.ac.uk/bioimaging/flim/" target="_blank">Cardiff Bioimaging</a> facility has a microscope that can perform these reactions so we offered to test their samples.</p>
  
 
<p>They provided bacteria containing three constructs that we grew overnight in the presence of 5uM copper sulphate -/+ 2mM Arabinose (which induced expression using the pBAD promotor).  
 
<p>They provided bacteria containing three constructs that we grew overnight in the presence of 5uM copper sulphate -/+ 2mM Arabinose (which induced expression using the pBAD promotor).  

Revision as of 14:00, 15 October 2016

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Collaborations


During our project we have had discussions with numerous teams, usually over Skpe or on twitter!

These discussions have involved teams based in:


> Washington University in St Louis, USA
> Paris-Sacley, France
> Oxford University, UK
> Trinity College in Dublin, Ireland

FLIM experiments with Oxford iGEM

The Oxford iGEM team wanted to test their copper chelators using Fluorescence Lifetime IMaging (FLIM), previous research by Hötzer et al (2011)had shown that GFP lifetime was modified in a copper concentration dependent manner. The Cardiff Bioimaging facility has a microscope that can perform these reactions so we offered to test their samples.

They provided bacteria containing three constructs that we grew overnight in the presence of 5uM copper sulphate -/+ 2mM Arabinose (which induced expression using the pBAD promotor).

These constructs were:

1. A constitutive GFP

2. Inducible CG:GFP cooper chelator protein (BioBrick Link

3. Inducible MG:GFP cooper chelator protein(BioBrick Link


With help of Dr Anthony Hayes in the Cardiff Biomaging facility we obtained the following FLIM data for their constructs. This has helped with their characterisation as described on their Wiki

GFP-Cu chelator FLIM experiment

Tables shows triplicate (i.e. 3 regions per cover-slipped area) values of the peak lifetimes resulting from measurements using Excitation=483nm, Emission=500-550nm.

Construct

Arabinose -ve

Arabinose +ve
Constitutive GFP Expression 2.6ns, 2.6ns, 2.6ns 2.6ns, 2.6ns, 2.6ns
pBAD-CG::GFP 3.3ns, 3.3ns, 3.3ns 2.5ns, 2.5ns, 2.5ns
pBAD-MG::GFP 2.6ns, 2.8ns, 2.6ns 2.2ns, 2.3ns, 2.3ns
Image below shows bacteria (A/C) expressing pBAD:MG-GFP grown -/+ 5mM Arabinose as well as graphs (B/D) showing fast lifetime values from 1000 measurements. Final values in the table above correspond to the most common value. Three measurements were taken from each sample.

ATP Measurements with Washington University, St Louis iGEM

Data to come...

Survey Participation

Cardiff_Wales