Lux pL controlled luxR with lux pR controlled GFP(lva tag).
This BioBrick system is able to notify the downstream activation of the lux R promoter of the 3OC6HSL Receiver Device.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device.
The lux p promoters originate from the quorum sensing ability of ''Vibrio fischeri''.
Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).
Lux pL controlled luxR with lux pR controlled AHL degrading enzyme AiiA(lva tag).
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. If 3OC6HSL is present in the cell, the autoinducer inactivation enzyme A (''aiiA''), with an LVA Tag, will inactivate the 3OC6HSL autoinducer molecules.
David Pohlman, Blythe Ferriere, Aile Able, Erica McFarland
Our part consists of an OMP-R binding region that regulates our reporter gene, eYFP. The OMP-R binding region is an omp-c operator, which has three OMP-R binding sites. Our part is regulated by the ENVZ-OMPR two-component regulatory system. ENVZ is a osmolarity sensor on the inner membrane and phosphorylates OMP-R in the presence of high osmolarity. When phosphorylated, OMP-R binds to the omp-c operator and recruits RNAP to transcribe the downstream reporter gene. Fluorescence intensity due to the production of eYFP is a measure of the system's activity. When all three binding sites are occupied by phosphorylated OMP-R proteins, RNAP cannot bind to the DNA and transcribe downstream. This part can be used as an osmolarity indicator. Our team used this part as a glucose sensor because the presence of glucose changes osmolarity. Different concentrations of glucose produce different, quantifiable fluorescence intensities.
Our team had to combine the genes in a specific order. First our team cut out and purified the eYPF gene. We added this gene to a plasmid containing RBS. Next we cut out the RBS+eYFP intermediate and ligated them into the plasmid containing OMP-R. The RBS and OMP-R parts were too small to run out and extract on a gel because they would run off of the end of the gel. Therefore, we had to start the process with eYPF.
This BioBrick was created from parts found in the Registry. These parts were BBa_R0082, BBa_B0032, and BBa_E0030.
AHL regulated PoPS generator: Las receiver device
Quorum sensing is the mechanism of communication between bacteria via synthesizing and detecting chemical compounds in the media. With these mediators, the promoters and their yield can be tuned or regulated. This part represents an alternative quorum sensing receiver device, which is able to generate a PoPS response in the presence of 6HSLO3 produced by LasI, instead of BBa_F2620. This will allow iGEMers to establish communications between different bacteria colonies and it makes simple to generate easy regulated complex systems. This part is assembled with Las promoter system to improve the current quorum sensing devices and parts which commonly base on Lux promoter system.
Note that pLas has no RBS afterwards, the assembling methods must be selected after this consideration.
pLas and LasR sequences are gathered from Registry.
This part is the one of the responding systems of the receiver device of our Photocommunication system
We used ncbi when we couldn't find sequence
EPIC Luciferase: BBa_K325100, Cambridge 2010 pOmpC: BBa_R0082
Barry Canton [bcanton@mit.edu] and Anna Labno [labnoa@mit.edu]
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the tet repressible R0040. Initial characterization work has been done on this device using I13273 as a test device.
Example Data Sheets for F2620
Lux pL controlled LuxR + lux pR promoter.
This part was made as a replacement for a defective version of BBa_F2621 which lacked the R0063 promoter, but was otherwise intact and functional. It shows a significantly higher response to 3OC6HSL than the incomplete BBa_F2621 (which is present in the Registry as BBa_K317037)
Was constructed by cloning BBa_R0063 in front of the incomplete BBa_F2621 (sent from the Registry in July 2011 lacking the R0063 subcomponent)
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the LuxR controllable R0063.
Lux pL controlled LuxR with lux pR autoinducing LuxI(lva tag)- AHL.
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. Complementary to the lux pL promoter, the lux pR promoter with a complex of luxR and 3OC6HSL will enable enlarged activation of the downstream synthetase gene, thus increase the concentration 3OC6HSL.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device. The resulting construct worked a lot better than the original F2621 - K082014 BioBrick system.
The lux p promoters and the autoinducer producing enzyme originate from the quorum sensing ability of ''Vibrio fischeri''.
This is the ''virG'' response regulator from the virA/G receptor system. The virA/G receptor system is used by ''Agrobacterium tumefaciens'' to detect phenolic substances secreted by plants. In presence of these substances, ''virG'' is activated by the ''virA'' receptor and induces the transcription of genes under the control of a ''vir'' promotor. Normally, ''virG'' needs the ''rpoA'' RNA polymerase subunit from ''A. tumefaciens'' to work in ''Escherichia coli'' but this ''virG'' BioBrick is mutated, so works with the ''rpoA'' subunit from ''E. coli''.
synthesized by Mr. Gene, so optimated codon usage, removal of illegal restriction sites
This device senses the N-butyryl-HSL concentration in the media and produces output PoPS. CinR production is controlled by the tet repressible R0040.
Pgad - Intracellular Chloride-Sensing Cassette
Pgad is chloride-sensitive promoter which was first discovered in Lactococcus lactis1, which is a gram-positive bacterium which can live in acidic environment. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of Pgad2. The gene before Pgad, named gadR, which is constitutively expressed under the control of PgadR, is a positive regulator of Pgad coupled genes while intracellular chloride is level elevated.
Synthesized part by GenScript,all the illegal sites for RFC 10 (EcoRI, XbaI, SpeI, PstI) have been substituted by other codon producing the same amino acid in E. coli.
The gene sequence was obtained from the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes PgadR, gadR, Pgad and the starting codon ATG.
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the lac repressible R0011.
This device senses the AI-1 concentration in the media and produces output PoPS. LasR production is controlled by the tet repressible R0040.
This device senses the 3OC14HSL concentration in the media and produces output PoPS. CinR production is controlled by the tet repressible R0040.
High Population->mRFP (pLac->LuxI; pLacIQ->luxR; pLux->mRFP;)
Quorum sensing circuit under a pLac promoter. Population turns red (mRFP) once quorum sensing kicks in.
Double terminator not used after LuxI because it is unnecessary and has the potential to make the plasmid more unstable.
Built through assembly of BBa_K084014 and BBa_J10000.