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 (Craney et al. 2007). The circuit is completed with a stop codon and a terminator sequence.

CHARACTERIZING THE SENDER CELL

We began our exploration of the sender-receiver system by examining three different sender systems:

1. HNS BW25113 Dhns::kan strain with lux biobrick. Because the H-NS protein has been shown to repress the Lux genes, we transformed the lux operon BioBrick (BBa_K325909) into this H-NS knockout E. coli strain.
2. NEB10 with lux biobrick. We chose to explore the light output of this strain as teams that have worked with the lux biobrick previous have chosen NEB10 as their chassis.
3. SY104. This strain contains a "split-lux" system to decrease the genetic payload controlled by a single promoter. Gene expression of lux AB is controlled by the sulA inducible promoter, and lux CDE expression is controlled by CP25 constitutive promoter.

We conducted at 16 hour time course at which we measured the light output of each of the aforementioned systems.

Figure 1. Normalized luminescence was calculated as a ratio of luminescence of a culture at a time point to OD₆₀₀ of the culture at that time point. Error bars represent standard deviation from the mean, and sample size was n=3 for all strains and time points.

The data demonstrated that

MATERIALS AND METHODS

Cultures of all three were grown to saturation, back diluted 1:100 , and, at an O.D.600 reading of ~.4, separated into twelve tubes (four per strain). Three tubes for each strain were induced with either arabinose for the lux operon BioBrick-containing H-NS and NEB10 strains and nalidixic acid for the SY104 sender strain. The final concentration of inducer was 10 mg/L for SY104 and .01 M for H-NS and NEB10. One tube per strain remained as an un-induced negative control. The luminescence in RLU’s with a 1000ms integration time and the O.D. at 600 nm was measured every two hours for sixteen hours after induction on a Tecan Infinite m200.