Difference between revisions of "Team:Cardiff Wales/Collaborations"
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| − | <p>Finally we participated with iGEM Toronto as one of the panel at a SynBio open panel event. This put our finished Human practices statement up to scrutiny, with discussions on the effectiveness of SynBio in LEDCs and how we can approach the introduction of our technologies into undeveloped areas.</p> | + | <p>Finally we participated with iGEM Toronto as one of the panel at a SynBio open panel event. This put our finished Human practices statement up to scrutiny from the general public, with discussions on the effectiveness of SynBio in LEDCs and how we can approach the introduction of our technologies into undeveloped areas.</p> |
<img id="prof" src="https://static.igem.org/mediawiki/2016/4/4b/T--Cardiff_Wales--tor1.jpg"/> | <img id="prof" src="https://static.igem.org/mediawiki/2016/4/4b/T--Cardiff_Wales--tor1.jpg"/> | ||
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Revision as of 09:03, 19 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:
> Oxford University, UK
> Washington University in St Louis, USA
> Paris-Sacley, France
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 copper chelator protein (BBa_K1980001)
3. Inducible MG:GFP copper chelator protein(BBa_K1980003)
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 |
The full analysis of these results can be found on the Oxford iGEM Wiki.
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. To increase ATP production they wanted to introduce phosphoenolpyruvate kinase (PCK) and phosphoglycerate kinase genes to E.coli. From us 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 coupled to a split reporter system. Additionally it later transpired that we both were going to use the E.coli codon optimised Cas9 from S.pyogenes, with additonal conserved mutation to knockout endonuclease activity. Because of this we were looking at potentially testing one an others guide constructs with our own reporters (Cardiff- Split Luciferase/ Saclay - Split GFP).
However both us and Saclay have found that we were unable to clone these dCas9's. This hints at an unforeseen compatibly issue arising either from the original biobrick or the mutations made to make into a dCas9. We speculate that the size of our constructs perhaps were unsuitable for plasmids we chose for cloning, or potentially too big for transformation. Unfortunately due to budget restraints we were unable to test electroporation to transform them which may have proved more successful.
Meetups
European Experience 2016 - Paris
Despite being newcomers to the competition we were eager to discuss and share our ideas with other teams. We first headed off to Paris barely 5 days after we finished training to present our design to over 20 different teams from 11 different countries. During the conference we became familiar with multiple teams, and received input into one of the potential designs of our system. This is where we discovered iGEM Paris Saclay - a fellow team using the same CRISPR-dCAS9 system.
iGEM Meetup - Westminster
Our second meetup was a more sober affair - 2 days of presentations from 16 other iGEM teams from across the UK. This was a chance to practice for Boston, and work in some last-minute changes into the project. This was in the last week of our main wet-lab phase, so by then we had wrapped up most of our work and were looking for ways to develop our presentational skills, as well as our Human practices and the potential design of our main project if taken past the proof-of-concept stage. In particular we discussed the viability of a paper-based system with iGEM Warwick
Toronto SynBio Panel
Finally we participated with iGEM Toronto as one of the panel at a SynBio open panel event. This put our finished Human practices statement up to scrutiny from the general public, with discussions on the effectiveness of SynBio in LEDCs and how we can approach the introduction of our technologies into undeveloped areas.
Survey Participation
