Luciferase from firyfly (Photinus pyralis). Firefly luciferase emits light by the oxidation of D-luciferin, its substrate. Luciferase can be used as a measuring tool when combined with other cis-elements (regions regulating the expression of genes on the same DNA molecule; promoters and other protein-binding domains), because of its highly quantitative performance.
RBS (medium) B0032, firefly luciferase CDS I712019, double-terminator B0014
VirA/G signaling system + sensitivity tuner + luciferase
This part combines a complete VirA/G signaling system with a sensitivity tuner before a luciferase gene under the control of a ''vir'' promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
sensitivity tuner to enhance the luciferase signal
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''virA'' and ''vir'' promoter from ''Agrobacterium tumefaciens'' C58, TI-plasmid
Protein coding region for the firefly [Photinus pyralis] luciferase, codon optimized for yeast.
luxABCDE gene ligated to the rear of T7 promoter. When transformed into BL21(DE3) E.coli strain, add different amount IPTG to reach different final concentration of IPTG in the medium can induce different level of Lux ligt emission. No substrate is needed.
luxABCDE gene ligated to the rear of T7 promoter. The original Ara and P-bad were replaced with T7 promoter, which enables its usage in our project.
T7 promoter and LuxCDABE with their RBS are from Partsregistry.
LuxCDABEG with corresponding six RBS(from K325909)
rLucN + ADH1 terminator + Kanamycin resistance
A luciferase from Promega's pGL4.10luc2 vector.
sequence of this vector is already published by Promega
Gluc coding device with Pr use as the positive control in gaussia luciferase assay
R.reniformis luciferase + ADH1 terminator + CaUra3
Bacterial Lux reporter CDEG pλ AB (E. coli optimised)
his is the first part of the split Renilla luciferase. To be used with fLuc[2].
A composite part consisting of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split fluorophor YFP with a fluolinker joined to its N-Terminus (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002 and the second half of split Luciferase For further information see the description of the parts mentioned above.
Rluc can express Renilla luciferase（Rluc）.
Rluc can express Renilla luciferase which has become popular as a reporter enzyme for gene expression assays. Renilla luciferase (RLUC)is a blue-light emitting luciferase of marine anthozoan Renilla reniformis [Str].As a reporter gene, rescarchers attach it to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. RLUC is chosen as a reporter because the characteristic it confer on organisms expressing it is easily identified and measured.The bioluminescence of the sea pancy,is under the control of a nerve network and is stimlated by changes of intracellular Ca??﹢ concerntramide，CO₂ andlight(λMax=480nm),as in the following scheme. RLBP is another protein of Renilla reniformis that can luminesce in vivo called Ca??﹢???triggered Renilla luciferin birding protein. 2015 SZU-IGEM construct Rluc with one codon being amber mutated and SV40 ,the promoter in the same plasmid to introduce Rluc into our system.This plasmid with Rluc,together with two other plasmids,are inserted into the cell.Only when the two promoters in plasmids without Rluc are activated simultanuously to express the downstrean DNA sequence can Rluc being translated completely. RLUC that is produced inside the cell catalyzes a reaction with luciferin to produce light. By measuring the light intensity using Luminometer,we can tell the working efficiency of our system under different conditions.
We designed the following primers and amplified Rluc promoter from the vector psi-Check2:Up: CCGGAATTCTCTAGACTGGCTGCCAAGTAGTAC Down: TGCACTGCAGACTAGTTACTGCTCGTTCTT. By incorporating these primers into Rluc promoter, the promoter is flanked by the iGEM prefix and suffix after amplification.
In our experiment, we got the part from Shenzhen Second People's Hospital. Additionally, we did our system's function verification in Shenzhen Second People's Hospital.
This page described the Lux operon from Vibrio fischeri. Lux gene is directly PCRed from the BBa_K325909, the pbad operon is deleted and now this part is available for any expression unit. You can provide light out put by putting this coding device under your promoter. We have successfully put this coding sequence into the PET28a expression system (cell strain is dH5α), and light out put could induced by the IPTG. To see more information about Lux light generator, please visit http://partsregistry.org/Part:BBa_K325909, and a more complete description can be found on the http://2010.igem.org/Team:Cambridge/Bioluminescence/G28.
In our project, we have to put the Lux light generator gene under the Ptet operon. So we have to relieve this part from the Pbad operon.
We use PCR extract the DNA sequence from BBa_K325909, and clone it into the PSB1C3.
vir promoter + sensitivity tuner + luciferase
This part combines a mutated virG response regulator from the VirA/G receptor system under the control of a constitutive promoter with a sensitivity tuner before a luciferase gene under the control of a vir promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''vir'' promoter from ''Agrobacterium tumefaciens'' C58, TI-plasmid
Second part of Split-Luciferase fused to the trans-membrane region of the Erbb1/EGF-receptor.
Derived by cloning of the parts BBa_K157002 and second part of Split-Luciferase. For further information see the description of the parts mentioned above.
Red Firefly Luciferase and LRE L. Cruciata (E. coli optimised)
This part is contains the coding sequence for a mutant light-emitting enzyme (luciferase) and luciferin regenerating enzyme (LRE) from the Japanese firefly, Luciola cruciata. It will make bacteria emit light at a reddish wavelength when placed under a promoter, provided the media is supplemented with luciferin. The luciferin regenerating enzyme will convert oxyluciferin into CHBT. CHBT is converted to more luciferin in the presence of D-cysteine.
Codon optimisation, making compatible with BioBrick assembly format.
This was codon-optimised for E.coli and syntesised by DNA 2.0.
Luciferin regenerating enzyme (L. cruciata)
Firefly luciferase (BBa_I712019) with 6X-His tagged for easier protein purification. Sequence was codon optimized by IDT codon optimization tools.
6X-His tagged for easier protein purification. Sequence was codon optimized by IDT codon optimization tools.
Rluc-linker-PEST191 is a part composed of Rluc (which acts as a reporter) linked with PEST191 through six aminoacid long protein linker. PEST191 is a sequence that leads towards degradation of protein linked with this sequence.
Rluc-linker-PEST 191 was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
Gaussia luciferase coding sequence optimized for use in E.coli DH5-Alpha
Gaussia luciferase coding sequence optimized for use in E.coli
luciferase: luciferin 2-monooxygenase from Renilla reniformis (EC 1.13.12.5; SwissProt:: P27652)
Rluc is a protein called Renilla`s luciferase and it emits light when adding the right substrate. That is why it can bi used as a reported to track other proteins or to monitor the activity of a promotor.
Rluc is cloned in BioBrick vector pSB1AK3.
This part combines a mutated ''virG'' response regulator from the VirA/G receptor system under the control of a constitutive promoter with a sensitivity tuner before a luciferase gene under the control of a ''vir'' promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
including N terminal 398 amino acid domain, 2??linker, and FRB.
N terminal 416 amino acid domain and FRB are linked with 2??linker.
medium strong constitutive promoter + luc
This is a firefly luciferase gene under the control of a medium strong constitutive promoter.
medium strong promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
* promoter and RBS from partsregistry * luciferase from pGL4.10luc2 (Promega)
Luminescence-responsive Promoter+GFP(BBa_E0840)
The luminescence-responsive promoter was engineered from promoter ColE7, during which the LexA binding site was mutated to guarantee that the endogenous LexA will not disturb the function of this promoter. This mutated LexA binding site within luminescence-responsive promoter can be recognized by corresponding mutated LexA DNA binding domain.
The LexA binding site was mutated to guarantee that the endogenous LexA will not disturb the function of this promoter. This mutated LexA binding site within luminescence-responsive promoter can be recognized by corresponding mutated LexA DNA binding domain.
This part contains both fragments of the split firefly luciferase, each of them preceded by an Elowitz RBS.
This is a firefly luciferase gene under the control of a strong constitutive promoter.
strong promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
This is the second part of the split Renilla luciferase. To be used with rLuc[1].
Protein coding region for the NanoLuc(tm) Luziferase (designed by the Promega Corporation, free for research use), Codon optimized for yeast. The Nanoluc is smaller than other luziferase proteins and yields equal or stronger luminescence readings.
gBlock designed by Team Tuebingen, sequence by Promega
luxC for light production protein generator
What does the device do? Helps to produce light What are the inputs and outputs to the device? Takes in a long-chain aldehyde + CoA + NADP(+) and produces a long-chain acyl-CoA + NADPH. Are any other devices needed? Also requires the other genes of the bacterial luciferase pathway. Does the device have any chassis dependencies?
This device uses a strong RBS to drive translation of luxC.
Uses part <partinfo>BBa_K424002</partinfo>
Firefly-renilla dual luciferase assay kit
Luciferase assay is one of the most popular reporter assay system for quantitatively measuring the function of promoters and other cis-elements. The wide-ranging and quantitative detection is the prominent feature of this assay; light emmision from 10~(-18) ― 10~(-11) mol protein can be quantitatively measured using luminometer (see our results). A promoter or other cis-element to be analysed can be ligated above this part. Its effect will be measured as an altered firefly luciferase expression. Meanwhile, renilla luciferase expression can be used to as an internal control.
including streptavidin, 2??linker, and firefly luciferase N terminal 416 amino acid domain.
streptavidin and N terminal 416 amino acid domain was linked by 2??linker
This is the nitrate- and nitrite-responsive promoter PyeaR of ''Escherichia coli'' with the luciferase of firefly ''Photinus pyralis'' (deposited by Slovenia iGEM 2007) with the addition of a strong bacterial ribosome binding site. It should generate light in the presence of nitrate/nitrite and luciferin.
No special considerations. Note that sequencing shows an unexpected insertion of 6 bases, ACCACC, after the scar between the ribosome binding site and the ATG of the luciferase coding sequence; that is, the sequence reads ACTAGACCACCATG rather than ACTAGATG. If this is correct, these 6 bases must be present in luciferase BioBrick BBa_I712019, or else were introduced by a random event during construction. This makes the RBS 12 bases from the ATG, somewhat more than the optimum, but with a strong RBS (like J15001) should still give about 25% of the expression level expected for optimum spacing in E. coli (Vellanoweth & Rabinowitz, 1992, Molecular Microbiology 6, 1105-1114).
Made by joining PyeaR BioBrick BBa_K216005 to a PCR fusion product of strong synthetic ribosome binding site J15001 and firefly luciferase BioBrick BBa_I712019.
Gaussia Luciferase (BBa_K1732003) with 6X-His tag for easier protein purification. Sequence was codon optimized for E.coli via IDT codon optimization tools.
6X-His tag for easier protein purification. Sequence was codon optimized for E.coli via IDT codon optimization tools.
This is a modified <partinfo>I746370</partinfo> sensitivity tuner. Instead of a GFP reporter, this one has a firefly luciferase (from Promega's pGL4.10luc2 vector) as one reporter gene. The arabinose promoter has been removed from the original part so now it is possible to assemble this part behind any promoter to get an enhanced luciferase signal.
arabinose promoter removed, GFP exchanged with luciferase compared to the original part <partinfo>I746370</partinfo>
* luciferase from pGL4.10luc2 (Promega) * the rest from the partsregistry
To edit. Pense ?? rajouter les linkers, je ne sais pas s'ils sont de igem ou s'il faut ajouter la s??quence ?? la registry. Check pour tous tes biobricks et dis-moi, please.
This part is the full renilla luciferase. The split protein has also been submitted as biobricks: BBa_K1486021.
LargeBit is the big part of a Split Luciferase. Its molecular weight is 18 kDa. This construct on its own has no function since LargeBit on itself has no luminescence activity. For luminescence activity, the SmallBit of the Split Luciferace is needed.
The given sequence from the company is used
NanoLuc (Nluc)is a novel engineered luciferase coupled with a novel coelenterazine analogue: Furimazine. When both are coupled, we obtain a much brighter luminescence than Fluc or Rluc and it is known to work exceptionally well as a reporter. NanoLuc is a 19.1 kDa enzyme which is an ATP independent luciferase providing a glow-type luminescence.
We have checked for all illegal restriction sites.
Kyle Hooper, Ph.D, Applications of a smaller, brighter, more versatile luciferase:NanoLuc??? Luciferase Technology, 2012
R.reniformis luciferase + ADH1 terminator + Kanamycin resistance
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''vir'' promoter from Agrobacterium tumefaciens C58, TI-plasmid
Fusion Protein of S-Layer SgsE and Firefly-Luciferase
This is the second part of the split Firefly luciferase. To be used with fLuc[1].
The aequorin-coelenterazine complex needs calcium to produce light. In the process this calcium enters through a special type of calcium channels present in the cell???s plasma membrane which open or close in response to a change in the transmembrane potential, that is why they are called voltage-dependent calcium channels (VDCC). The equation which describes calcium current through these channels is the following: Where g is the conductance associated with the channel, V is the transmembrane potential and ECa is the Nernst potential, related to the different ionic concentration inside and outside the cell. Considering that these channels are only permeable to calcium and have two states -open or closed-, the total conductance associated with the population of VDCCs can be expressed as the maximal conductance ( ) times the fraction of all channels that are open. This fraction is determined by hypothetical activation and inactivation variables m and h, which depend on voltage and time: VDCCs are well characterized in excitable cells such as neurons, where the value of is known: Budding yeast, Saccharomyces cerevisiae has homologous voltage-dependent channels in its plasma membrane. However, there is a lack of study of the properties of these channels. That is why we have determined their conductance according to our experimental results: Having characterized calcium entry to the cell, we studied the aequorin production of light through a calcium-mediated response after two types of stimulation: ??? Chemical stimulation Alkali shock (performed by the addition of KOH) induced a calcium entry to cytoplasm (where aequorin is located) through voltage-dependent calcium channels. These calcium ions bind the aequorin-coelenterazine complex and photons ???light- are emitted during a short period of time. This graph shows yeasts??? response to the addition of 30 ??L of KOH: Furthermore, we made experiments with increasing KOH volumes (in microliters) and realized that light emission was higher: the more KOH arrives to cells, the more calcium ions get into the cytoplasm and more photons are emitted by aequorin. It has to be noted that, in presence of suitable amounts of coelenterazine, the aequorin production of light can be repeated with several stimulations of 30 microliters each (where arrows show): Finally, we have proved that calcium enters through VDCCs, as yeast strains lacking this calcium channels (cch1 and mid1) emit less amount of light. Two negative controls (addition of EDTA, a quelant of calcium ions, and KCl, which do not perform alkali shock) are also included in this graph: ??? Electrical stimulation Calcium entrance to cytosol ???and the consequent aequorin light emission- can also be perfomed by applying electricity to the yeast culture. This response was characterized at different voltage values and times of application. Again, the stimulation was repeatable (stimulus applied where arrows): *The following graph show data obtained with an ELISA plates reader which made discontinuous measures, one each 30 s. Therefore, we had not information about light production between dots. The time of exposure to the electrical stimulus was crucial, even more that the aplied voltage. That means, if we increased the voltage at very short times, cells could produce a more abrupt peak of light. But if we increased the time of exposure to the electricity, we observe a less defined response, with more flattened peaks. That???s probably because a big exposure time of electrical input damages and kills the yeasts, making them to release their components to the medium, including the aequorin-coelenterazine-Ca2+ complex, so the emission of light is more uniform in time, instead of the production of the flash produced by the Calcium entering in the cell. In the case of very little voltages (like 1,5V) this observation is not carried out by our yeasts. The reason must be that the electrical input is too low, so yeasts don???t die so easily as with more elevated voltage, and a better response is produce with a more prolongated electrical shock. Although the previous graphs show that the second stimulation induces a lower amount of light, varying the time of stimulus application, higher peaks could be obtained: Similarly to chemical stimulation, yeast mutant strains lacking calcium channels had less light emission. Several negative controls (water, SD medium, SD with coelenterazine without yeasts, yeasts without coelenterazine, and addition of EDTA) were made to prove that light came from aequorin-transformed yeasts: *The following graphs show data obtained with a continuous luminometer. It has to be noted the sharp peak of light produced when stimulation is applied (we were not able to detect it with the discontinuous measurements) In the following graphic WT aequorin-transformed yeasts and mutant strains (lacking cch1 and mid1) are compared again. SD medium with coelenterazine and yeasts without coelenterazine are included as negative controls: Again, light production is lower in mutant cells, also in the first peak of light -that we were not able to see with the discontinuous luminometer-, as calcium ions enter through VDCCs. A sharp peak of light is produced when the stimulus is applied, but our controls discard the idea of an artifact. For example, light could be made by a spark produced during the discharge. It is not very probable, because the peak observed was produced during near 400 seconds after the stimulus. Another time, when cells without coelenterazine or mutants were used, we saw no light.
It comes from the genomic sequence of the luminiscent jellyfish Aequorea victoria.
Part consists of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split fluorophor YFP with a fluolinker joined to its N-Terminus (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and Part one of Split- Luciferase For further information see the description of the parts mentioned above.
Celia Chenebault, Camille Soucies, Benjamin Piot
The Pr promoter is a constitutive promoter driving the transcription of the XylR gene, coding for the XylR protein. The XylR protein, a transcriptional regulatory protein for the Pu promoter, is activated by aromatic hydrocarbons that carry a methyl group (like toluene and xylene). In our biosensor the Pu promoter allows the transcription of the bioluminescent reporter gene GLuc, coding for the Gaussia luciferase. When this enzyme reacts with its coelenterazine substrate it emits luminescence.
Site directed mutagenesis to overcome the mutation in the start codon of XylR
IDT synthesis in 3 parts that were then assembled
Microbes including Escherichia coli are known to respond to various DNA-injuring stress (ionizing radiation, ultraviolet radiation, peroxides etc...), altering their gene expressions. This response, known as "SOS response", are induced by regulatory protein called RecA, which is activated when binds to single-strand DNA. DNA-RecA complex promotes the self-degradation of LexA, a common repressor for SOS genes. SulA is responsible for stress-induced halt of cell division. The promoter of sulA, or sulAp, is acutely induced by various stress factors, including ultraviolet irradiation. We provide a sulAp evaluation device to make it easy to measure the varying expression of sulAp in various "stressful" conditions. Employing a dual luciferase reporter construct, quantitative measurement and comparison of Relative Promoter Unit (RPU) is achieved.
sulAp + dual luciferase reporter construct
sulAp (BBa_K518010) was cloned from Escherichia coli K12 strain's genome.
This BioBrick is for testing the virA/G signaling system. The read-out is the luciferase <partinfo>K389004</partinfo>. This system contains an unmutated ''virA'', so it is possible to measure, how the natural virA/G system works and reacts.
this is only for measuring the natural virA/G system
''A. tumefaciens'' C58 TI-plasmid, partsregistry, Mr. Gene, Promega's pGL4.10luc2 plasmid
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor), which was our main target for modifying the signaling path. dnMyD88 is linked to Rluc (which acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-Rluc was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
Nluc luciferase is an enzyme that was engineered to work as a luminescent reporter weighing 19.1 kDa. This protein uses fumarizine as novel substrate to produce a high intensity glow-type luminescence. These reactions are ATP-independent, trying to obtain the maximal assay sensitivity. Reference: [1] Promega (2015). Nanoluc Genetic Reporter Vectors. URL: http://worldwide.promega.com/products/reporter-assays-and-transfection/reporter-vectors-and-cell-lines/nanoluc-luciferase-reporters/nanoluc-genetic-reporter-vectors/
For our project purposes its use is destined to work as a reporter of protein expression
This is a firefly luciferase gene under the control of a weak constitutive promoter.
weak promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
This part encodes a reporter cassette for the Cre Recombinase (e.g parts XYZ) in Yeast. An active Cre protein will switch expression from RFP to NanoLuc luciferase.
bacterial luciferase LuxAB of Xenorhabdus luminescens
This part includes the coding sequences of ''luxA'' and ''luxB'' of ''Xenorhabdus luminescens'', with their native ribosome binding sites. The dimeric bacterial luciferase LuxAB produces light in the presence of oxygen, FMNH2, and a long chain aldehyde. The aldehyde can be provided by the ''luxCDE'' gene products, but if these are not present, n-decanal can be added to cultures.
No special considerations. Please note that preliminary sequencing suggests that there may be a problem with the SpeI site of the construct submitted. If this is confirmed, we will submit a revised version and remove this note.
Amplified from genomic DNA of ''Xenorhabdus luminescens''.
Part 1 of the Split- Luciferase fused to the trans-membrane region of the Erbb1/EGF-receptor.
Derived by cloning of the parts BBa_K157002 and Split-Luciferase fragment 1. For further information see the description of the parts mentioned above.
dnMyD88-linker-Rluc-linker-PEST191 is a part where dnMyD88 (very important protein in signaling cascade through TLR4 receptor) is linked to Rluc (reporter) and PEST191 (degradation tag). Both linkers are made of six aminoacids.
dnMyD88-linker-Rluc-linker-PEST191 was made by using PCR ligation technique. It was difficult since we had some problems with primers. This part is cloned in BioBrick vector pSB1AK3.
coding sequence of the fusion protein of CFluc of the luciferase and the dCas
This part contains both fragments of the split renilla luciferase, each of them preceded by an Elowitz RBS.
With this system it is possible to measure the activity of a ''virA'' receptor and the ''vir'' promoter, respectively. The reporter is a firefly luciferase from Promega's pGL4.10luc2 plasmid.
medium strong constitutive promotor, terminator to keep basal expression of luciferase low
partsregistry, synthetic gene by Mr. Gene, pGL4.10luc2 by Promega, TI-plasmid from ''Agrobacterium tumefaciens'' C58
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002 and first half of split Luciferase For further information see the description of the parts mentioned above.
Firefly luciferase from Photinus pyralis
Chris French and William Galdric Rostain
In luminescent reactions, light is produced by the oxidation of a luciferin (a pigment): luciferin + O2 → oxyluciferin + light Thus luciferase is a a reporter protein when connected to other proteins or promoters prodicing light, by the oxidation of substrate luciferin. This is derived from BioBrick I712019, but PCR was used to remove the extra bases before the start codon (these are shown in the Registry sequencing data but are not indicated in the sequence as entered by Ljubljana 2007; presumably they are there to make a proper eukaryotic context for the start codon). This BioBrick contains the bare coding sequence in pSB1C3.
Forward sequence looks good. Reverse sequence missing the base between the stop codon and SpeI site. This is also missing in the S284T clone (see below) suggesting that it is missing in the primer, or did we use a vector based primer? In which case it would suggest that it was missing in the original clone. Trivial but annoying.
The origional sequence is from Photinus pyralis, a the gene here is derived from BioBrick BBa-I712019.
including FKBP, 2??linker, and firefly luciferase C terminal 394 amino acid domain.
FKBP and C terminal 394 amino acid domain are linked by 2??linker
C394 terminal domain of firefly luciferase
This is a modified <partinfo>BBa_I746380</partinfo> sensitivity tuner. Instead of a GFP reporter, this one has a firefly luciferase (from Promega's pGL4.10luc2 vector) as one reporter gene. The arabinose promoter has been removed from the original part so now it is possible to assemble this part behind any promoter to get an enhanced luciferase signal.
arabinose promoter removed, GFP exchanged with luciferase compared to the original part <partinfo>BBa_I746370</partinfo>
rLucC + ADH1 terminator + CaUra3 cassette
Gaussia Luciferase, also known as Gluc, is a bioluminescent luciferase protein that was isolated from Gaussia princeps. When reacted with the optimal concentration of the substrate coelenterazine, a green light will luminescence.
This gene has been codon optimized for E.coli.
Designed by Cheryl Telmer of the Bruchez Lab in Carnegie Mellon University.
This is the first part of the split Firefly luciferase. To be used with fLuc[2].
This is nitrate- and nitrite-responsive promoter PyeaR attached to bacterial luciferase luxAB of ''Xenorhabdus (Photorhabdus) luminescens''. It produces light in the presence of nitrate or nitrite when supplied with a long chain aldehyde (eg, decanal) as substrate.
Made by joing BBa_K216005 to BBa_K216008
This is the tightly regulated lac operon <partinfo>K389050</partinfo> with a luciferase gene as readout.
lacIq integrated in promoter so good regulation, luciferase as sensitive readout
This is a modified <partinfo>BBa_I746390</partinfo> sensitivity tuner. Instead of a GFP reporter, this one has a firefly luciferase (from Promega's pGL4.10luc2 vector) as one reporter gene. The arabinose promoter has been removed from the original part so now it is possible to assemble this part behind any promoter to get an enhanced luciferase signal.
This is nitrate- and nitrite-responsive promoter PyeaR of ''Escherichia coli'' joined to ''Renilla'' luciferase with the addition of a strong bacterial ribosome binding site. This should lead to production of light in the presence of nitrate/nitrate and coelenterazine.
No special considerations. Sequencing suggests that there is an extra 'A' base following the scar between the RBS and the Renilla luciferase; that is, the scar region reads TACTAGAATG instead of TACTAGATG. If the sequencing is correct, this base must be present in J52008 or have been introduced by a random event during the construction process. This makes the gap between the RBS and start codon one base longer, but still well within the optimal range for E. coli and B. subtilis.
Made by joining PyeaR BioBrick BBa_K216005 to a PCR fusion of strong synthetic ribosome binding site J15001 and Renilla luciferase BioBrick J52008.
Fusion Protein of S-Layer SbpA and Firefly-Luciferase
This is a optimized firefly luciferase (C-terminal modification, see design notes).
Primers were designed to replace the C-terminal SKL aminoacids with KL.
The original source is the Firefly luciferase, see http://www.ncbi.nlm.nih.gov/nuccore/AB644228.1 The modification is covered by 'One-step measurement of firefly luciferase activity in yeast' (http://www.ncbi.nlm.nih.gov/pubmed/14558144).
Firefly luciferase - luciferase from Photinus pyralis
Firefly luciferase is luciferase from Photinus pyralis. By oxidation of substrate luciferin light is produced. Luciferase thus acts as a reporter protein when connected to other proteins or promoters.
Part is cloned into pSBAK3 vector with terminator.
This firefly luciferase comes from Promega PGL3 vector.
This version of the LuxC comes from Vibrio salmonicida. Team Example used it to build a bacterial lava lamp.
No modifications were necessary in order to comply with the standard.
Vibrio salmonicida hypothetical protein EJN1 gene, complete cds; luminescence gene cluster, complete sequence; and RibG (ribG) gene, complete cds. NCBI Accession number is AF452135 REGION: 2346..3776
SmallBit is the small part of a Split Luciferase. Its molecular weight is 1 kDa. This construct on its own has no function since SmallBit on itself has no luminescence activity. For luminescence activity, the LargeBit of the Split Luciferace is needed.
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and Part 2 of Split- Luciferase For further information see the description of the parts mentioned above.
Outer Membrane Protein X (OmpX) with BsoBI-linker and NanoLuc
OmpX is an outer membrane protein with the C- and N-termini in the intracellulair domain. To be able to use OmpX as a scaffold, a non-natural amino acid needs to be introduced. This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in, which can be used for the SPAAC click chemistry reaction with DBCO functionalized groups. The BsoBI linker is a 213 bp long flexible GGSGGS linker. Using the restriction enzyme BsoBI, the linker can become 45 bp shorter. NanoLuc is a small sized and bright luciferase. Under the addition of furimazine, the NanoLuc luciferase generates a bioluminescece emmission spectrum with a maximum around 460 nm.
The sequence is redesigned considering codon optimalization. Also restriction sites for biobricking and classical cloning are deleted. From the BsoBI linker, the coding sequecne for GGSGGS is redesigned causing that only two BsoBI restriction sites remain.
OmpX is a outer membrane protein naturally occuring in wildtype E.coli. The BsoBI linker is inspired by the article "Quantitative Understanding of the Energy Transfer between Fluorescent Proteins Connected via Flexible Peptide Liners" by Toon H. Evers et all from 29 August 2006. NanoLuc naturally occurs in the organism Oplophorus gracilirostris and was optimalized by the company Promega.
LuxC gene from V. fischeri for generating light.
Which species is the enzyme from? Vibrio fischeri What reaction does it catalyze? One of five steps in the production of light. Does it work in E. coli? Yes, several groups have used this operon as a reporter in E. coli. Does it require any other parts? Yes. The full luxCDABE operon is needed for light production.
Requires the other four steps in the LuxABCDE process
ACCESSION Y00509 REGION: 1624..1866 AUTHORS Engebrecht,J. and Silverman,M. TITLE Nucleotide sequence of the regulatory locus controlling expression of bacterial genes for bioluminescence JOURNAL Nucleic Acids Res. 15 (24), 10455-10467 (1987) PUBMED 3697093
This part combines a complete VirA/G signaling system with a sensitivity tuner before a luciferase gene under the control of a vir promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''virA'' and ''vir'' promoter from Agrobacterium tumefaciens C58, TI-plasmid