Lux pL controlled LuxR + lux pR promoter.
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 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)
Promoter (luxR & HSL regulated -- lux pR)
Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
Promoter (luxR & HSL regulated -- lux pL)
The lux cassette of V. fischeri contains a left and a right promoter. The left promoter gives weak constitutive expression of downstream genes.This expression is down-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription from Pr, repressing transcription from Pl
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below.Includes most of Lux reulatory region, including the LuxR binding site which activates the right promoter. A putative LuxR autorepression binding site is also identified adjacent to the -10 site of the right promoter. This 2nd site has 55% identity with the first site. Putative inverted repeats (of size 18-27 bp) also exist between these two sites (not marked above), which may represent binding sites for other regulatory proteins. <img src="<bb_file>Image01.gif</bb_file>" width="614" height="362"> Unspecified.
This device senses the 3OC6HSL concentration in the media and regulates the expression of downstream genes. The complex of the LuxR activator protein and the 3OC6HSL binds to a palindromic site on the promoter, increasing the rate of transcription.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006 and Part BBa_K118005.
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs) and Part BBa_K118005 (CrtI with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006 and BBa_K118005 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274110) and arabinose-inducible promoter I0500 (see Part BBa_274120), transformed and tested in E.coli strain MG1655. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that the Composite Biobrick, when put under a promoter, works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (lycopene) production than strain TOP10.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
V. fischeri <genbank>AF170104</genbank>
In complex with HSL, LuxR binds to the Lux promoter, activating transcription from Pr <bb_part>BBa_R0062</bb_part>, and repressing transcription from Pl <bb_part>BBa_R0063</bb_part>. The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux activator, LuxR complexed to HSL. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
2 silent point mutants were introduced in the coding sequence to remove internal XbaI and PstI sites. Mutation sites were chosen to replace codons commonly used in E. coli with codons used at a similar frequency.
Double terminator consisting of BBa_B0010 and BBa_B0012
Synthetic ribosome binding site added by Son of Babel procedure
This is a 4bp synthetic ribosome binding site which can be added by fusion PCR using a reverse primer and the Son of Babel cloning system.
The part can be added by fusion PCR using a Son of Babel reverse primer.
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
crtE (geranylgeranyl pyrophosphate synthase) coding sequence.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis parthway.
crtB coding sequence encoding phytoene synthase
This is the coding sequence of crtB from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene synthase, part of the carotenoid biosynthesis pathway, which converts geranylgeranyl diphosphate to phytoene. Reference: Misawa,N., Nakagawa,M., Kobayashi,K., Yamano,S., Izawa,Y., Nakamura,K. and Harashima,K. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172 (12), 6704-6712 (1990)
crtI coding sequence encoding phytoene dehydrogenase
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene.
The native crtI sequence contains two internal PstI sites. Primers crtImutf1 and crtImutf2 were ordered so that the MABEL protocol (invented for this purpose) could be used to remove these sites. The mutagenic primers were as follows: * crtImutf1: gtt tac agt aag gtt gcc * crtImutf2: agt aac tct ctg ttt gtg c In both cases, the PstI site CTGCAG was converted to CTGCAA, a silent mutation.
Transcriptional terminator consisting of a 64 bp stem-loop.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Conversion of the iGEM parts registry to SBOL2.1