Difference between revisions of "Team:Penn/Sender"

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<p>We began our exploration of the sender-receiver system by examining three different sender systems:</p>
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<p class="margin-top-10">Our sender cell characterization was founded on determining the <b>photons/second trends</b> 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.</p>
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<li><strong>HNS BW25113 Dhns::kan strain with lux biobrick.</strong> 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.</li>
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<p class="margin-top-10">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.  
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<li><strong>NEB10 with lux biobrick</strong>. 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.</li>
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<li><strong>SY104</strong>. 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.</li>
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<p class="margin-top-10">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 substrate externally to the reaction and concentrating cell samples to increase light output. The project plan is illustrated in the flow diagram below</p>
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<p>We conducted at 16 hour time course at which we measured the light output of each of the aforementioned systems.</p>
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<p class="margin-top-10"><br><b></b> </p>
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<p><em>Figure 1. </em>Normalized luminescence was calculated as a ratio of luminescence of a culture at a time point to OD<sub>600</sub> 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.</p>
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<p class="margin-top-10"><br><b>DERIVATION OF PHOTONS/SEC FROM RELATIVE LIGHT UNITS (RLUs)</b> </p>
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<p class="margin-top-10"> Our team used a Tecan M200 <insert specifications here> in order to measure luminescence output of our cell cultures. The instrument provides the luminescence in relative light units. We worked to convert this to an absolute measurement, which would allow us to compare our luminescence output to pDawn (our receiver plasmid) activation thresholds described in literature.
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<p class="margin-top-10"> The below graphs demonstrate the derivation of our conversion. The first graph relates RLUs to luminometer measurements. A luminometer is "an instrument used to measures light and other optical properties of specimens in chemiluminescent and bioluminescent applications." Following this, the second graph relates the luminometer measurements to power meter readings. The luminometer is used as an intermediate because the low intensities of the cell cultures are below the detection threshold of the power meter.</p>  
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Revision as of 01:13, 19 September 2015

University of Pennsylvania iGEM

PENN iGEM 2015



SENDER


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.

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.