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Novel bioluminescent reporters

Finding, testing and generating optimised bioluminescent reporter vectors for use in various organisms.

The Idea

Bioluminescent reporters are a common tool in molecular biology. Luciferases from various sources (firefly, click beetle, dinoflagellate, sea pansy, copepod and bacterial) have been cloned and exploited as reporters, due to their fast time dynamics and their detectable and quantifiable outputs. Each luciferase has particular enzymatic properties and substrate requirements, leading to limitations on how assays can be performed. While all eukaryotic luciferases require the addition of an exogenous substrate (eg. luciferin, calcium ions) to produce light, bacterial bioluminescence is generated by a single lux operon which has the benefit of autonomous luminescence [Engebrecht et. al, Cell 1983].

Yet, compared to fluorescent protein reporters, little engineering has been applied to improve and optimize the lux operon on a protein level. Our idea is to sequence the genomes of various wild free-living marine bioluminescent bacterial species, in order to discover novel genes and gene variants responsible for bioluminescence and use these as novel luminescent reporters.

Seminal research has demonstrated important aspects about the structure of the lux operon in bioluminescent bacteria and has highlighted fundamental regulatory elements for light production

[Bassler et al., Mol Microb. 1993; Fuqua et al., J Bacteriol. 1994]. Recently, the 2010 Cambridge iGEM team provided an excellent demonstration of the use of synthetic biology practices to implement lux operon derived bacterial bioluminescence in E. coli. The team expressed a codon­optimized lux operon cassette driven by the arabinose inducible pBAD promoter, free of the endogenous quorum sensing regulation, and they were able to achieve high levels of tunable bioluminescence.

Although research has focused on symbiotic bioluminescent bacteria, many free­living marine bacteria exhibit bioluminescence to a highly variable degree. Whereas some strains can be seen only after several minutes in a dark room, bright strains are distinguishable by a blue glow even in low light levels. Bacterial bioluminescence has been studied since the late XIX century [Beijerinck, Arch Neerl des Sci Exact et Nat 1889], however, there have been few attempts to systematically quantify the luminosity of free­living environmental bioluminescent bacterial strains and search for the underlying genetic differences that might account for this variance of luminosity. Our plan is to systematically study a collection of such bacteria that have been gathered and isolated from marine environments around the world, in order to gain insight into their light producing mechanisms and generate reporters.

After sequencing the genomes and finding the sequences of bioluminescent genes, we plan to test them in a standardised E. coli context, in order to gain insight into the structure­function relationships. On the basis of this data, we will then generate optimised bioluminescent reporter vectors for use in various organisms.

 

The Team

Bernardo Pollak
Anton Kan
Anton Kan

 

 

 

 

 

 

 

Bernardo Pollak 

Graduate Student, Department of Plant Sciences

Anton Kan 

Graduate Student, Department of Plant Sciences

 

 

Project Outputs

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The Synthetic Biology Strategic Research Initiative provides a hub for anyone interested in Synthetic Biology at the University of Cambridge, including researchers, commercial partners and external collaborators. 

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