This project would build on our previous work identifying and optimizing bacterial luciferases from a variety of marine bacteria. We propose to generate versions of these reporters for eukaryotic systems, including the production of Nanolantern-like systems for lux luciferases.
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 generated by the lux operon 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.
Recently, development of the Nanolanterns [Takai et al. 2015] from Renilla luciferase variants has expanded the colour palette for luciferase reporters, as well as enhancing their brightness. This development enables real-time multi-channel luminescence measurements which can have significant applications for cell biology and gene expression analysis. These use reporters are also somewhat limited through their requirement of the addition of coelenterazine substrate.
The bacterial luminescence pathway has have been inserted into eukaryotic systems previously, with some success in yeast and mammalian systems [Close et al. 2010]. Often the luminescence yield is limited compared to the bacterial context due to the difficulty of expressing polycistronic operons in eukaryotic systems. In plant systems, previous studies have only inserted the luxAB luciferase, which would requires the addition of decanal as a substrate [Cui et al. 2014].
This project would build on our previous work identifying and optimizing bacterial luciferases from a variety of marine bacteria. Our work as part of the April 2015 SRI’s funding call has led us to generate a variety of autonomous lux operon reporters and luciferase reporters through directed evolution. In order to develop these reporters further for eukaryotic systems, we propose to generate versions of these reporters for eukaryotic systems, including the production of Nanolantern-like systems for lux luciferases. This will allow the use of highly efficient autonomous luminescence production for eukaryotic systems with multiple colors, which will provide new systems for gene expression and cell biology studies, as well as any other applications where light energy needs to be produced from chemical energy.
We will use the brightest evolved proteins obtained from our previous SRI project to design and synthesize codon-optimised versions of these lux reporters for a plant chassis. In our previous work, we reorganised the lux operon (luxCDABEG) into the luciferase component, luxAB, and the substrate pathway component, luxCDEG. To reengineer the reporters for the plant context, we will redesign the polycistronic substrate pathway operon into simple monocistronic transcriptional units. Recent work on Marchantia polymorpha has also discovered and tested a range of native promoters that can be used to carefully tune the expression of each of the components to their optimal levels. We will also design and generate monomeric versions of the luxAB reporter, and through translational fusions to fluorescent protein expanding of the colour palette for multi-channel imaging and detection. Due to the complexity of the genetic reorganisation, DNA synthesis provides a very attractive route to the generation of such constructs. We will proceed to test these systems in Marchantia polymorpha to demonstrate the use of bright and autonomous lux reporters in eukaryotic systems.
Benefits and outcomes
Our project aims to develop a range of novel bright, autonomous, multi-channel bioluminescent reporters, as well as vectors for their expression in eukaryotic plant systems. These will be made publicly available.