IS THE LIGHT PRODUCED BY THE SENDER CELL SUFFICIENT TO ACTIVATE THE RECEIVER CELL?

INTRODUCTION

An effective light-based communication system rests on the bioluminesence generated by the “sender cell.” In order to design a well-functioning system, the Penn 2015 iGEM team worked to optimize the light output of various E.coli “sender cells” transformed with the lux operon (BBa_K325909).

Lux operon expression is responsible for bioluminescence. The operon is initiated by a constitutive promoter (BBa_J23100) followed by an RBS + lux box. The box contains the following: LuxC, D, A, B, E and G. LuxA and B encode two subunits of bacterial luciferase. The genes LuxC, D, and E drive expression of the substrate for the light-emitting reaction, tetradecanal. The function of the luxG gene is yet to be fully elucidated; however, inclusion of the gene is known to increase light output (CITATION). The circuit is completed with a stop codon and a terminator sequence.

Our sender cell characterization was founded on determining the photons/second trends for luminescing cell populations. This information was important in order to determine if light produced by the lux box is sufficient to activate the light-activated transcription factor of the receiver cell population. Following this, we measured luminescence output in different strains (BL21, NEB10 and HNS BW25113 Dhns::kan strain). We wanted to see which strain would drive the most sustained luminescence output at the highest photon/second value. We also tested the light output of a "split-lux" system to decrease the genetic payload controlled by a single promoter. Gene expression of lux AB was placed under control of the sulA inducible promoter, and lux CDE expression was placed under a constitutive promoter (CP25/CP38). Further characterization involved adding more substrate to the reaction and concentrating cell samples to increase output. The project plan is illustrated in the flow diagram below