During our project we have been in communication with numerous teams.
These discussions have lead to collaborations with the team from:
> 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)
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.
|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 WashU_StLouis iGEM
Over the past few years iGEM teams from WashU in St Louis have 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 (PGK) genes to E.coli. From us they wanted some sensitive supplementary analysis of ATP synthesis by their PCK and PGK constructs.
WashU sent us two plasmids to us for test using an ATP-Luciferase Assay with our FLUOROstar Optima luminometer.
We transformed these plasmids to obtain E.coli that contained eith pBAD-PCK or pBAD-PGK and then followed this protocol:
> E.coli were grown o/n. Subcultured 1/10, grown for 2hr before arabinose (at 25mM, 200uM, 1.6uM) was added for 5hr. This was repeated in triplicate.
>Centrifuge cultures, resuspend in 100ul PBS. Add 900uL 0.1M Tris pH7.5, 2mM EDTA. Boil for 5minutes, centrifuge to remove cell debris. Freeze samples.
Luciferase reactions were set up as follows:
>Reaction buffer: 50ul Luciferase, 50ul 600uM luciferin, 50ul sample (containing known [ATP] or 50ul PCK/PGK sample)
>Measure 10 times and add values for the total light output value for each sample.
>Take average from 3 replicates for a final value.
This data reveals that higher levels of PCK appears to increase ATP concentrations within the samples. However there are a number of caveats attached to these results. Firstly the light measurements were below the values obtained in the ATP standard curve and secondly the 'PCK 0' measurement had a higher than expected value.
However although these results need to be repeated, they largely confirm what the WashU team had observed in their own analysis.
Exploring dCas9 with Saclay
We met members from Team Saclay at the European Meetup. After discussing our projects we recognised that we both planned on utilising dCas's specific DNA binding function coupled to a split reporter system. Additionally it later became clear that we both were going to use the E.coli codon optimised Cas9 from S.pyogenes, with additonal conserved mutations to knockout endonuclease activity. This linkage inspired us to plan to look at potentially testing each others guide constructs with the reporters we had individually developed (Cardiff- Split Luciferase/ Saclay - Split GFP).
However both teams were unfortunately unable to clone this dCas9 gene. This is due to unforeseen issues arising either from use of the original biobrick or from our cloning schemes. From the Cardiff perspective we speculate that the size of our construct (approximately 5kb) made this experiment unsuitable for the cloning technique we had initial chosen, which is outlined on our Parts page. Ultimately it was useful to discuss the similarities in our project and swap war-stories about the techniques that we might use for cloning these genes.
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.