This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the lux operon from Vibrio fischeri.

• Description
• Arabinose to light
• H-NS mutants

Description

E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)

This page described the lux operon from Vibrio fischeri. To relieve LuxR control we placed Lux C, D, A, B, E under the pBad promoter.

Figure 1 - E.coli cells (Invitrogen TOP 10) transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] in a 96 well plate.

Arabinose to light

This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below.

Data

Figure 1 - Light output of <partinfo>K325909</partinfo> as a function of Arabinose concentration in the media. The values correspond to the peak intensity within 5 hours of adding Arabinose to the media. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats.

Figure 2 - Evolution of luminescence with time at different Arabinose concentrations for <partinfo>K325909</partinfo>. Measurements were taken every 30 minutes. Data points and error bars correspond to the mean and standard deviation of 3 time repeats.

H-NS mutants

It has been shown that the expression of the Vibrio fischeri lux operon when cloned into E. coli was repressed. This repression was linked to the nucleoid protein H-NS. To investigate this effect we cloned the operon into mutant E.coli cells in which the expression of the H-NS protein had been modified. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below.

Data

Figure 1 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.

Figure 2 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10 with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.

Figure 3 - Figure 3 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan]. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.

Figure 4 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.

Effect of pH

Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below.

Data

Maximum light output within 5 hours of D-luciferin injection at different pH values.

[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]

These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.

Evolution of light output at different values of pH.

[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]

Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.

Protocol

1. The protocol can be found as Experiment 110.

Compatibility

[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell Chassis:] Device has been shown to work in Top 10 (Invitrogen) Plasmids: Device has been shown to work on <partinfo>pSB1C3</partinfo>

References

[http://www.ncbi.nlm.nih.gov/pubmed/18949818 [1]:] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,Life 61, 6-17.

[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html [2]:] T. Nakatsu et al. (2006) Structural Basis for the spectral difference in luciferase bioluminescence, Nature 440(16), 372-376.

[http://www.ncbi.nlm.nih.gov/pubmed/11457857 [3]:] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, The Journal of Biological Chemistry, 276(39), 36508-36513.