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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:

> 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 cooper chelator protein (BBa_K1980001)

3. Inducible MG:GFP cooper 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

Image below shows bacteria (A/C) expressing pBAD:MG-GFP grown -/+ 2mM 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.

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

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. *description here*

Our second meetup was a more sober affair - 2 days of presentations from 16 other iGEM teams from across the UK. This was nearer to the end of our main wet-lab phase, so whilst we had much more to present, the event itself allowed for much more scrutiny than Paris. etc.