Introduction

What is bioluminescence ?
Bioluminescence is visible light emission in living organisms mediated by an enzyme catalyst. The bioluminescence has been observed in many different organisms including bacteria, fungi, fish, insects, algae, and squid.(1)

lux system
Lux operon codes for bacterial luciferase subunit (luxAB) and the fatty acid reductase polypeptide (luxCDE) that is responsible for the biosynthesis of the aldehyde substrate for luminescent reaction.
The luxA and luxB gene code for α and β subunit of luciferase. Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide (FMNH2) and long-chain fatty aldehyde resulting in the emission of a blue-green light.(1)

FMNH2 +O2 → FMN + RCOOH + H2O + light

The luxCDE genes are required for conversion of fatty acid into long chain aldehyde.
luxC : reductase
luxD : transferase
luxE : synthase

the luxG gene which codes for flavin reductase is very closely linked to the luxE gene


advantage of lux system
Adding substrate is NOT necessary
Light emission occurs AUTOMATICALLY


Objective
1. Cloning lux operon from Photobacterium phosphoreum
Why we chose lux gene from Photobacterium phosphoreum?
P. phosphoreum (NCMB844) is known that the luminescence intensity is brightest of other strains. (2)
But maximum luminescence levels require lower temperatures than the other luminescent species, because this species live in the deeper waters of the ocean. (2)
Method
Construction of biobrick
Original lux gene from P. phosphoreum has three biobrick restriction sites.


In order to remove restriction site, we chose overlap extension PCR method and designed four primer set.


Lux gene fragment was amplified and four PCR products were connected.


First, we constructed arabinose inducible lux gene biobrick.
Evaluation method
We first investigate the best culturing condition.
Transformed E coli. TOP10 was precultured into LB medium and incubated at 37℃. After incubating for 12 hour, precultured solution was inoculated into 100 mL LB medium. When OD660 of the culture became 0.6, arabinose(0.25% f.c.) was added to induce and expressed the lux proteins at different temperature(20 ℃ and 30 ℃). After induction, the 500 μL of culture was harvested at certain time and measured light intensity and OD by plate reader.

Fig. Investigation of culturing condition

Result

Here, we show the result. It is suggested that at lower temperature(20 ℃), lux operon from P. phosphoreum kishitanii can emit brighter light than at 30 ℃. However, since we first measured the intensity of the light at 3 hours after induction, there is still possibility that the E. coli cultured at 30 ℃ emitted brighter light within 3 hours. To check the change of light intensity within 3 hours from induction, we operated another experiment. In this time, we measured the intensity every 30 minutes until 3 hours.


The figure below shows the result of second experiment. As we supposed, the intensity of the light emitted by E. coli cultured at 30 ℃ came to its peak within 3 hours. Yet, the peak intensity was much lower than that of 20 ℃. Then we concluded that 20 ℃ is better to express this lux operon.

Reference (1) Meighen EA. (1993) Bacterial bioluminescence: organization, regulation, and application of the lux genes The FASEB Journal vol. 7 no. 11 1016-1022 (2) Edward A. Meighen Autoinduction of light emission in different species of bioluminescent bacteria Luminescence 1999;14:3–9 2. Improvement of lux luminescence

We succeeded in constructing arabinose inducible lux gene biobric and found out the suitable culturing condition to express, so we next tried to improve the coli express system by changing the color, strengthening the light intensity, and shortening the reaction time.

Change the color
We used LumP, YFP, and GFP proteins to change the color.
What is LumP and YFP ?
Bacterial luciferase derived from Vibrio fischeri, which is a strain of luminous bacteria, shows brue-green light emission (wavelength is 495 nm).
However, Vibrio fischeri strain Y-1 emits longer wavelength light than normal Vibrio fischeri (535 nm, yellow)(3). The yellow shift of light emission wavelength is caused by YFP, which is a secondary emitter protein. YFP does not emit light itself, but it interacts with the luciferase, resulting in a shift of wavelength of light emission. YFP has FMN as a chromophore, and its size is about 28 kDa.
Furthermore, in luminous bacteria, there are strains that emit light with shorter wavelength than normal Vibrio fischeri (475 nm,blue), such as Photobacterium phosphoreum, Photobacterium kishitanii and Photobacterium leiognathi.
It is known that blue shift of these luminous bacterium is caused by lumazine protein (LumP). Similar to YFP, LumP shifts the wavelength by interacting with the luciferase. As a chromophore, it has 6,7-dimethyl-8-(1'-D-ribityl)lumazine (DMRL), and its size is about 21kDa.

Learning from previous reports, we decided to use these proteins for changing the color.

Reference
(3) Daubner SC, Astorga AM, Leisman GB, Baldwin TO., 1987, Proc Natl Acad Sci U S A.

Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein.


Method

Construction of biobrick
First, we produced the constitutive promoter + lumP + double terminator construct.

Then, we combined the construct to the construct of cloned Lux operon with Pbad promoter.

The construct of Lux operon with luxY is produced by joining the Lux operon and luxY biobrick part.



BRET
Bacterial luciferase from Lux operon emits light by catalyzing oxidization of luciferin. Oxidized luciferin, oxiluciferine, has high energy. When the oxiluciferin transits from high energy state to low energy state, light is emitted.
On the other hand, fluorescence substance, such as GFP, absorbs the light and becomes high energy state. And when the fluorescence substance transits from high energy state to low energy state, light is emitted.


If the luciferase exists near the fluorescence substance, Bioluminescence Resonance Energy Transfer (BRET) occurs. BRET is a phenomenon that can be observed when a fluorescence substance is excited by obtaining energy from the bioluminescence. This only happens when the wavelength of bioluminescence is the same as the wavelength that excites the fluorescence substance.

The wavelength of bacterial luciferase from Lux operon (497 nm) is close to the wavelength that GFP excites(501 nm).
We considered that BRET might occur between the bacterial luciferase and the GFP, so we designed the construct of Lux operon with GFP.

Method
Construction of biobrick

The construct of Lux operon with GFP is produced by joining the Lux operon and GFP biobrick part.

Evaluation method
Transformed E coli. TOP10 was precultured into LB medium and incubated at 37℃. After incubating for 12 hour, precultured solution was diluted with LB medium to the point that OD660 became 0.6, then applied into 96well microtiter plate. After adding arabinose(0.25% f.c.), the plate was shaken at 20 ℃ to express the lux proteins. The spectrum of the light and intensity was measured by plate reader once in an hour.

Result
1.lux+LumP
We could observe the shift of wavelength from 488 nm to 478 nm (Figure.a). The changing color can clearly recognize by the photograph(Figure.e). And we also achieved to enhance the light intensity. Maximal absorption of Lux+LumP is about 3-fold, comparing to that of lux(cloned).
Furthermore, we demonstrated that the lumP part(Edinburgh 2009, BBa_K216007) could work with the Lux operon from Photobacterium phosphoreum kishitanii strain.


[Figure.a]

1.lux+GFP

[Figure.b]

1.lux+YFP

[Figure.c]

4.Comparing to lux from BioBrick part(Cambridge 2010, BBa_K325909)

[Figure.d]


[Figure.e]Left is lux+GFP, middle is lux(cloned), right is lux+LumP.



Strengthen the intensity
Rib operon
P. phosphoreum has rib operon(ribEBHA) downstream of luxG. Their gene productshave been identified as riboflavin synthase(RibE), DHBP synthase(RibB), lumazine synthase(RibH) and GTP cyclohydrolaseII(ribA).
Riboflavin is the key substrate for lux operon because riboflavin is the precursor of riboflavin 5'- monophosphate (FMN). FMNH2 is the substrate for emitting light. Synthesis of riboflavin is important to enhance the luminescent. Instead of rib operon from P. phosphoreum, we cloned the rib genes from E. coli BL21(DE3) and constructed rib operon biobrick.



shorten the reaction time

Method
Construction of biobrick

Evaluation method
Transformed E coli. TOP10 was precultured into LB medium and incubated at 37℃. After incubating for 12 hour, precultured solution was inoculated into 100 mL LB medium. When OD660 of the culture became 0.6, arabinose(0.25% f.c.) was added to induce and expressed the proteins at 20 ℃. After induction, the 500 μL of culture was harvested at every 30 minutes and measured light intensity and OD by plate reader.
Result




As we expected, when only luxA or luxD were induced, the light intensity reached its peak faster than wild type. However, the maximum intensity of these two construct far lower than wild type. It may be caused by loss of mass balance of lux proteins. We could improve the light emission by expressing luxAB or luxCDE together not only luxA or luxD.

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