Difference between revisions of "Team:Cardiff Wales/Collaborations"
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<p>We met Saclay at the European Meet Up. Where upon discussions of our projects we recognised that we both where planning on utilising dCas's specific DNA binding function. Additionally it later transpired that we both were going to use the E.coli codon optimised <a href="http://parts.igem.org/Part:BBa_K1774001">Cas9</a> from S.pyogenes with conserved mutation to knockout endonuclease activity. Both us and Saclay have found that we were unable to clone these dCas9's, perhaps hinting at an unforeseen compatibly issue arising either from the original biobrick or the mutations made to make into a dCas9. | <p>We met Saclay at the European Meet Up. Where upon discussions of our projects we recognised that we both where planning on utilising dCas's specific DNA binding function. Additionally it later transpired that we both were going to use the E.coli codon optimised <a href="http://parts.igem.org/Part:BBa_K1774001">Cas9</a> from S.pyogenes with conserved mutation to knockout endonuclease activity. Both us and Saclay have found that we were unable to clone these dCas9's, perhaps hinting at an unforeseen compatibly issue arising either from the original biobrick or the mutations made to make into a dCas9. | ||
Revision as of 13:01, 16 October 2016
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. Therefore, it was hypothesised that if the chelators where functional then the lifetime of the fused GFP's would drop. The Cardiff Bioimaging facility possesses a confocal laser scanning microscope with FLIM module so we offered to test their samples.
Oxford sent us three cultures containing 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 |
ATP Measurements with Washington University, St Louis iGEM
Washington 2016 have for a number of years been investigating methods to transfer the nitrogen fixing ability of cyanobacteria Synechocystis into E. coli. This year they are looking at increase electron donor and ATP production with the idea to improve nitrogenase activity. They wanted some sensitive supplementary analysis of ATP synthesis by their phosphoenolpyruvate kinase (PCK) and phosphoglycerate kinase (PGK) constructs. Washington sent these constructs to us for testing by ATP-Luciferase Assays with our Biospace Lab PhotonIMAGER Optima.
Data to come...
Dodgy dCas9 with Saclay
We met Saclay at the European Meet Up. Where upon discussions of our projects we recognised that we both where planning on utilising dCas's specific DNA binding function. Additionally it later transpired that we both were going to use the E.coli codon optimised Cas9 from S.pyogenes with conserved mutation to knockout endonuclease activity. Both us and Saclay have found that we were unable to clone these dCas9's, perhaps hinting at an unforeseen compatibly issue arising either from the original biobrick or the mutations made to make into a dCas9.
Survey Participation
