"
Team:Tokyo-NoKoGen/Project/lux operon
From 2012.igem.org
Tokyo-NoKoGen
2012
- overview
- lux operon
- rhodopsin
- modelling
- Future work
- project
lux operon
The luxA and luxB 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 are required for conversion of fatty acid into long chain aldehyde.
luxC : reductase
luxD : transferase
luxE : synthase
the luxG which codes for flavin reductase is very closely linked to the luxE gene
Light emission occurs AUTOMATICALLY
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)
Original lux gene from P. phosphoreum has three illegal 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 tried to construct arabinose inducible lux gene biobrick.
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
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.
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.
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.
Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein.
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.
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.
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]
2.lux+GFP
We observed the maximal absorption of Lux+GFP at 512 nm (Figure.b). We thought that the shift of emitting light wavelength was caused by BRET. After 1 hour culture, peak could not be observed at 512 nm, and we performed the culture in dark condition. So it is possible to occur that GFP absorbes the lux luminescence and emits fluorescence.
[Figure.b]
3.lux+YFP
We could not observe the yellow light from lux+YFP construct (Figure.c). We assumed that the yellow shift didn’t occur due to difference of luminous bacteria strain (YFP is from Vivrio fischeri.).
[Figure.c]
4.Comparing to lux from BioBrick part(Cambridge 2010, BBa_K325909)
We also evaluated the difference of the light emission property between our cloned lux operon and lux operon from BioBrick part(BBa_K325909) (Figure.d). Sequentially, it was revealed that our lux and Cambridge lux had almost same maximal absorption peak and the light intensity.
.jpg)
[Figure.d]
[Figure.e]Left is lux+GFP, middle is lux(cloned), right is lux+LumP.
Strengthen the intensity
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 (luxAB) and the fatty acid reductase complex (luxCDE) that is responsible for the biosynthesis of the aldehyde substrate for luminescent reaction.The luxA and luxB 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 are required for conversion of fatty acid into long chain aldehyde.
luxC : reductase
luxD : transferase
luxE : synthase
the luxG which codes for flavin reductase is very closely linked to the luxE gene
advantage of lux system
Adding substrate is NOT necessaryLight 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 illegal 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 tried to construct arabinose inducible lux gene biobrick.
Evaluation
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 and discussion
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;14:3–92. 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
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]
2.lux+GFP
We observed the maximal absorption of Lux+GFP at 512 nm (Figure.b). We thought that the shift of emitting light wavelength was caused by BRET. After 1 hour culture, peak could not be observed at 512 nm, and we performed the culture in dark condition. So it is possible to occur that GFP absorbes the lux luminescence and emits fluorescence.
[Figure.b]
3.lux+YFP
We could not observe the yellow light from lux+YFP construct (Figure.c). We assumed that the yellow shift didn’t occur due to difference of luminous bacteria strain (YFP is from Vivrio fischeri.).
[Figure.c]
4.Comparing to lux from BioBrick part(Cambridge 2010, BBa_K325909)
We also evaluated the difference of the light emission property between our cloned lux operon and lux operon from BioBrick part(BBa_K325909) (Figure.d). Sequentially, it was revealed that our lux and Cambridge lux had almost same maximal absorption peak and the light intensity.
[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.
Method
Construction of biobrick
We cloned ribA, ribB, ribC, ribF, and ribDH from E.coli BL21(DE3).
Original rib genes, ribA and ribDH, have biobrick one restriction site in each genes.
In order to remove restriction site, we chose inverse extension PCR method.
After sequencing of each gene, we started to construct rib operon. First, ribosome binding site (RBS) was ligated with each gene. Second, each genes are ligated. Finally, promoter and double terminator were ligated. We constructed rib operon biobrick.
Result
coming soom...
Reference
(4) Nack-Do Sung and Chan Yong Lee. The lux Genes and Riboflavin Genes in Bioluminescent System of Photobacterium leiognathi are under Common Regulation Journal of Photoscience (2004), Vol. 11(1), pp. 41-45
shorten the reaction time
Method
Construction of biobrick
Pconst-lux CDBFEG –DT (deletion of luxA) was constructed by inverse PCR.
Primers with restriction site EcoRI and XbaI at 5’ end and SpeI and PstI at 3’ end was used to clone luxA
Arabinose inducible promoter was added in front of luxA.
These two parts were connected.
The same process was performed to construct this part.
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.
-
-
-
-
-
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.
Method
Construction of biobrick
We cloned ribA, ribB, ribC, ribF, and ribDH from E.coli BL21(DE3).Original rib genes, ribA and ribDH, have biobrick one restriction site in each genes.
In order to remove restriction site, we chose inverse extension PCR method.
After sequencing of each gene, we started to construct rib operon. First, ribosome binding site (RBS) was ligated with each gene. Second, each genes are ligated. Finally, promoter and double terminator were ligated. We constructed rib operon biobrick.
Result
coming soom...Reference
(4) Nack-Do Sung and Chan Yong Lee. The lux Genes and Riboflavin Genes in Bioluminescent System of Photobacterium leiognathi are under Common Regulation Journal of Photoscience (2004), Vol. 11(1), pp. 41-45shorten the reaction time
Method
Construction of biobrick
Pconst-lux CDBFEG –DT (deletion of luxA) was constructed by inverse PCR.
Primers with restriction site EcoRI and XbaI at 5’ end and SpeI and PstI at 3’ end was used to clone luxA
Arabinose inducible promoter was added in front of luxA. These two parts were connected.
The same process was performed to construct this part.
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.
