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 previously 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.Trend lines for each strain were created using MATLAB's curve fitting tool. All points after the peak luminescence were modeled using the exponential equation f(x)=a*exp(b*x) + c*exp(d*x). Points leading up to peak luminescence were modeled with a linear equation since due to low resolution. For coefficients see the materials and methods section included at the bottom of the page.

From these key take away points multiple suggestions about the future usage of these strains can be made. In fact, these three strains can be split into two categories: (1) Sustained Expression, and (2) Transient Expression.

SUSTAINED EXPRESSION – SY104:

The most noteworthy observation of SY104’s expression of bioluminescence is its extremely long sustained expression. As can be seen, up to 16+ hours the cultures being tested were still producing luminescence. This type of behavior though desired by many, as it provides simpler modeling and predicting of behavior using external light as a substitute, is only advantageous in specific systems. More specifically, such prolonged expression is only beneficial in a sender-receiver circuit when the receiver is extremely sensitive and does not reach its half-saturation point very quickly. In other words, this type of receiver is capable of being receptive to the low levels of luminescence produced in the earlier stages after induction. Since the time it takes to reach half-saturation point is slower than other receivers, this also means that it must be exposed to light for a longer amount of time in order to achieve its full potential. Thus, making SY104 a perfect match for a receiver of this sort.

TRANSIENT EXPRESSION – HNS & NEB10:

HNS and NEB10 exhibit behavior quite opposite of SY104. Both strains demonstrate a rapid peak in luminescence and then rapidly decay approximately one log order. The important observation in this trend is that the peak is actually significantly higher than any value that SY104 reaches. This is the reason why such strains as the HNS and NEB10 can be useful in sender-receiver systems where the receiver has an extremely high half-saturation time and is less sensitive. With such a receiver it is important not only quickly pack it with light but to also pack it with a high enough intensity of light that it will actually activate it. This is specifically the reason why a sender demonstrating sustained expression is not ideal in this case scenario, as it provides a significantly low amount of luminescence in early stages after induction.

Note that these differences between the strains does not necessarily mean that one is simply better than the others. Instead, this data supports the uniqueness of each strains characteristics and allows for reasonable arguments to be established about when to use one strain versus another.

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.

Coefficients for Best Fit Models