The structure of toehold switch is similar to hairpin, except it has a loop at the top as ???toehold???. Toehold switch functions as riboregulator through linear-linear interaction between RNAs. When target RNA appears, it will bind one of the toehold switch stems and open the loop, exposing the RBS. Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the gene being regulated. Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 50 end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single-stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the gene of interest. Our toehold is upstream of T3 RNAP. The trigger RNA is miRNA-144.
The toehold loop can be opened by trigger RNA, thus controlling the expression of T3 RNAP.
position 3&5 mutated promoter lux pR-3/5 (luxR & HSL regulated)
The position 3&5 mutated promoter lux pR-3/5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-3/5 gives a lower expression of downstream genes in an autoinduction system. In another word, the lux pR-3/5 has a lower efficiency than the lux pR(BBa_R0062). Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
We have generated two point mutants by site-directed mutagenesis from the sequence of lux pR(BBa_R0062). In these mutants, the G residue of position 5 was changed to a C and the C residue of position 3 was changed to a T respectively.
Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator
Siyang HAO, Tong MU, Zheng LUO, Mengqi ZHANG
This is a GFP generator with a theophylline responsive riboswitch sequence. The mRNA with a theophylline riboswitch in it would respond to theophylline concerntration, producing different amount of GFP. Under the control of different promoters, it could be used to demonstrate the theophylline responsive curve, providing data for modeling. The theophylline riboswitch sequence could also be used to regulate the translation of other genes.
The RBS of this device is altered into AGGAGGU. When changing the RBS, the anti-RBS sequence should be changed as well.
Theophylline riboswich is composed of a synthesized theophylline aptamer and a regulatory platform.
Theophylline responsive riboswitch Theo 1G1
Taken from no other than Curious George, this promoter is induced by the presence of banana in the metabolism of any mammal. It is very weak however without the presence of the HngR protein. For transcription to reach significant levels both HngR and banana must be present
Monkeys can be dangerous when aggrevated
This E. coli promoter is positively regulated by arabinose. araC is the regulator protein, binding to arabinose prevents the repression of pBAD.
This part is similar to BBa_I0500, but it differs from that one in several point mutations in the sequence.
Cup-1 is a regulatory promoter that senses heavy metals such as copper, arsenic, cadmium, antimony, mercury, chromium, and selenium.
Comes from genomic DNA of saccharomyces cerevisiae.
pspoIIE promoter (spo0A J01004, positive)
promoter from b. subtilis, activated by spo0A
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
AND-type promoter synergistically activated by cI and CRP
OmpF promoter that is activated or repressesed by OmpR according to osmolarity.
Promoter OmpF is activated by phosphorylated OmpR at low osmolarity, and is repressesed by phosphorylated OmpR at high osmolarity.In Escherichia coli, osmoregulation is mediated in part by the actions of such a two-component system consisting of EnvZ and OmpR. These proteins act to control the relative levels of the outer membrane porin genes, ompF and ompC. At low osmo larity, OmpF predominates in the outer membrane, while at high osmolarity the OmpF porin is replaced by OmpC.OmpF has a larger pore and a faster ??ow rate than OmpC. Reference: K. Mattison, R. Oropeza, N. Byers, L. J. Kenney, J Mol Biol 315, 497 (Jan 25, 2002).
The osmolarity range of OmpF sensing still have to be confirmed
Promoter OmpF is activated by phosphorylated OmpR at low osmolarity, and is repressesed by phosphorylated OmpR at high osmolarity.In Escherichia coli, osmoregulation is mediated in part by the actions of such a two-component system consisting of EnvZ and OmpR. These proteins act to control the relative levels of the outer membrane porin genes, ompF and ompC. At low osmo larity, OmpF predominates in the outer membrane, while at high osmolarity the OmpF porin is replaced by OmpC.OmpF has a larger pore and a faster ??ow rate than OmpC.
This part is the region starting from 537bp upstream of ChnB to its start codon from Acinetobacter sp. cyclohenol gene cluster. pChnB is activated by ChnR (K1946000) in the presence of cyclohexanone. Iwaki H, Hasegawa Y, Teraoka M, Tokuyama T, Bergeron H, Lau PC. Identification of a transcriptional activator (ChnR) and a 6-oxohexanoate dehydrogenase (ChnE) in the cyclohexanol catabolic pathway in Acinetobacter sp. Strain NCIMB 9871 and localization of the genes that encode them. Appl Environ Microbiol. 1999;65(11):5158-62. Steigedal, Magnus and Svein Valla. "The Acinetobacter Sp. Chnb Promoter Together With Its Cognate Positive Regulator Chnr Is An Attractive New Candidate For Metabolic Engineering Applications In Bacteria". Metabolic Engineering 10.2 (2008): 121-129. Web.
The exact region where ChnR binds is not known.
This part is from the first 537bp of GenBank: AB006902.2
Zif268-HIV bs + LexA bs + mCYC promoter
The Zif268-HIV and LexA transcription factor binding sites on the mCYC minimal promoter (in that order.) Transcription regulation comes from Zif268-HIV and LexA-binding transcription factors.
Constructed in our lab with parts from Caroline Ajo-Franklin and David Drubin.
This is the promoter activated by the transcriptional regulator XylR (BBa_I723136) in response to detection of environmental xylene.
Modified lambda Prm promoter (OR-3 obliterated)
Lambda Prm promoter modified to be activated but not repressed by the lambda repressor (cI)
In wild type lambda phage, the OR3 site (-27 to -11) reads "tatcccttgcggtgata" on the sense strand of DNA. To prevent lambda repressor (cI) from binding to this site, the 4th through 10th nt of OR3 were replaced by the 7 nt between OR2 and OR1, "aaatagt" (-57 to -51), which in effect mutates 5 nt of the promoter. These nt were chosen to be about halfway between the -10 and -35 boxes of the promoter. Furthermore, since these nt already act as a spacer in wild-type phage, it is hoped they will not create undesired interactions.
This cold shock promoter is only active at temperatures lower than 30 degree Celsius.
This is a modified K112400 heat shock promoter in BBa standard.
Fabiana de Melo Duarte, Leonardo Minete, Maria Carolina Grassi and Lucas P. Parizzi
Py is the tra operon promoter of F-plasmid. It is repressed by the host nucleoid-associated protein, H-NS and activeted by TraJ protein. Conflicting evidence suggests that TraY can act as both an activator and a repressor. The tra operon encodes F-plasmid proteins (except TraJ and TraM) involved in DNA transfer by conjugation in E.coli. The regions shows intrinsic curvature, which could interfere in regulation. For this reason, the two closer bends upstream promoter region are included.
The mechanism for controlling transcription from Py remains undefined. The Py region contains significant intrinsic curvature, which could interfere in regulation
Arabinose induced promoter. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Regulated by the presence of Iron and UV light irradiation
Promoter driving the rhlI gene in Pseudomonas aeruginosa. Activated by the RhlR+C4HSL. Also likely activated by LasR+3OC12HSL
Genomic sequence of Pseudomonas aeruginosa
Rock-paper-scissors - Paper primary plasmid
Composite part, consisting of BBa_K180005 and BBa_K180015, but with BBa_K180005 reading along the reverse strand, and BBa_K180015 reading along the forward strand. Forms the 'paper' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180005 reading along reverse strand, and BBa_K180015 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180005) has no effect over GFP or RhlI expression in BBa_K180015.
See relevant biobrick component parts (BBa_K180011 and BBa_K180012).
LasR and 3OC12HSL regulated promoter with a single Tet Operator (binding site for TetR repressor)
PAI+LasR -> LasI+GFP & AI+LuxR --| LasI+GFP
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the policistronic expresion of LasI enzyme and GFP, double terminator
The biobricks BBa_K266005 and BBa_E0840 where used to build this biobrick.
Right facing promoter (for xylA) controlled by xylR and CRP-cAMP
removed RBS cut out several xylR activation sites
CRP-cAMP transcriptional dual regulator activates transcription
XylR-Xylose transcriptional activator activates transcription
SPI-1 TTSS secretion-linked promoter from Salmonella
This promoter is activated when functional SPI-1 type III secretion systems are present and functional in Salmonella typhimurium 1344.
The promoter includes the binding site for the transcription factor InvF. This transcription factor is activated by the SicA chaparone when the secretion system is active.
This part was cloned from Salmonella typhimurium 1344 genomic DNA.
promoter for FabA gene - Membrane Damage and Ultrasound Senstitive
According to Vollmer, et. al in their 1998 Applied and Enviormental Microbiology paper, "Bacterial Stress Responses to 1-Megahertz Pulsed Ultrasound in the Presence of Microbubble", the promoter for the fabA gene, a known membrane damage response gene, is upregulated seven fold when cells are exposed to high frequency ultrasound and microbubble cavitation.
Rock-paper-scissors - Scissors primary plasmid
Composite part, consisting of BBa_K180018 and BBa_K180019, but with BBa_K180018 reading along the reverse strand, and BBa_K180019 reading along the forward strand. Forms the 'scissors' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180018 reading along reverse strand, and BBa_K180019 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180018) has no effect over mBanana or LuxI expression in BBa_K180019.
See relevant biobrick component parts (BBa_K180018 and BBa_K180019).
Promoter (lambda cI and luxR regulated -- hybrid)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
cI repressor negatively regulates this promoter and LuxR activates its transcription.The effect of cI is dominant over LuxR. This part is based on the LuxR and cI repressor regulated hybrid promoter designed and tested by Ron Weiss. It requires the binding of two cI repressor dimers for maximal repression and contains two cI repressor binding sites namely, OR1 and OR2. This promoter is leaky in the sense that 'some' transcription is seen in the absence of both cI and LuxR. LuxI cI activity of promoter + + zero + - maximum - + zero - - leaky (no quantitative information)
This part was designed based on the LuxR and cI repressor regulated hybrid promoter tested by Ron Weiss and the LuxR-LuxICDABE sequence annotated by Tom Knight AF170104 .
This gene encodes the terephthalic acid(TPA) transporting protein. TPA is not a kind of nutrient substance for most organisms, including common chassis organisms like E.coli and Saccharomyces cerevisiae. However, as a kind of TPA degradable organism, RhodococcusjostiiRHA1 can transport TPA into their cells because of the TPA transporting protein genetpaK in their genome. This gene is also verified to be useful in Saccharomyces cerevisiae.
It comes from the genome of RhodococcusjostiiRHA1.
Rock - Primary plasmid (part 2) [tac pR, mCherry and LasI generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'rock' primary plasmid.
Regulated expression of LasI (C0178) and mCherry (K180008) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180008, B0015)
Promoter from the copper-sensetive CusR/CusS two component signal system. (E.Coli)
Sequence from the NCBI database, with added BioBricks prefix and suffix.
ftsAZ promoter is a promoter with levels of expression are inversely proportional to the growth rate. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0021/al0022 on MG1655 (804bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112320 ----------------------------------------------- al0021 Forward Biobricking of ftsAZ promoter CCATAGAATTCatgAGATCTaacgacagtgttggtgagcg al0022 Reverse Biobricking of ftsAZ promoter CGttaGGATCCgtaccaatctccagtcctactaccag
Pm is the middle promoter of the Mu bacteriophage. It is in charge of the transcription of the activator protein C. Pm is activated by a product of the early transcription, protein Mor. It can be used to build a sensitivity tuner alongside this protein, this construct can be used to increase the amount of POPS in a certain genetic construction.
Synthetized from the reported sequence of Pm promoter from Mu bacteriophage.
FliA is a transcription factor involved in the formation of the flagella in E. coli. The promotor of fliA is induced by FlhDC. FliA seems to have a positive autoregulation on its promotor.
This part comes from the genome of E. coli K12 strain MG1655.
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of rfp gene, which together is connected to vector PSB1C3 afterwards, and therefore we are able to observe the OmpR promoter efficiency through the control of light.
We constructed this biobrick through pcr.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompF. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes three OmpR binding sites: F1, F2, and F3. The promoter starts at -107 and goes up to the transcriptional start site at +1. A distant fourth binding site, F4, has been deleted. It is involved in negative regulation of ompF (Head, Tardy and Kenney, 1998), so its deletion is intended to make the promoter solely activating. The efficacy of this promoter versus the ompC upstream region (BBa_R0082) or the truncated ompC upstream region (BBa_R0083) is currently unknown.
Rock-paper-scissors - Scissors secondary plasmid [las pR, LacI generator]
iGEM2009 Team:Southampton rock-paper-scissors 'scissors' secondary plasmid.
Regulated expression of LacI (C0012) by Las pR (R0079).
See relevant basic biobrick parts (R0070, B0034, C0012, B0015).
This promoter is the right promoter in the lux cassette of Vibrio Fischeri. There is weak constitutive expression of downstream genes. Expression of downstream genes can be up-regulated by the action of the LuxR activator protein in complex with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein can form a complex with two molecules of the AHL. This causes a conformational change in the protein, allowing it to bind to a site on the lux promoter and induce transcription.
This part was obtained by taking the reverse complement of R0062, ordering appropriate oligos, then annealing and extending them.
This is the same part as BBa_K110007 except it has Prefix and Suffix regions switched
This is the whole regulatory region from the URA3 gene coding for OMP decarboxylase in S. cerevisiae budding yeast.
NCBI Sequence database: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=Nucleotide&dopt=GenBank&val=172061
Promoter of flhB gene from E. coli flagella
''flhB'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCACGTCATATCAGGCGGTCTGATAAGG *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTTTGTCGTCGCTCTCGTCAGACACGTC
This part comes from the genome of ''E.coli'' K12 strain MG1655.
Intermediate designed to accelerate cloning of the HSP promoter ( BBa_K338001 ) by adding an RBS downstream.
Designed to be ligated upstream of the desired heat-shock product. Keep in mind that the promoter is somewhat leaky and that cultures should be grown at 30°C.
The Registry of Standard Biological Parts
This is the PF promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PF promoter.
no special considerations seemed necessary
The original source is the P2 phage genome. For our work the DNA came from plasmids, kindly supplied to us by Prof. Calendar, University of California (Berkeley): Bacteriophage PSP3 and fR73 Activator Proteins: Analysis of Promoter Specificities; Julien and Calendar, 1996; JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5668???5675
An acid inducible promoter of the acid shock response (asr) gene, extracted from E. coli K12. This part can be used to generate pH based response in any biological system.
The sequence provided in literature indicated a consensus sequence for the Pho Box just before the transcription start site
This part was extracted from the genome of E. coli K12
This promoter was isolated by taking 500bp upstream of the MF(ALPHA)2 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
This gene is on the crick strand of the entered sequence.
This device is regulated by UV irradiation
promoter to xylA without CRP and several binding sites for xylR
removed crp binding site removed rbs removed several xylR binding sites
GoL - Primary plasmid (part 1)/RPS - Paper primary plasmid (part 1) [LuxR generator]
Part of the iGEM2009 Team:Southampton Game of Life primary plasmid.
Constitutive promoter selected to allow for continuous expression of C0062.
AcrAB-TolC multidrug efflux transport system activator from E. coli
The MarA protein acts as a transcriptional activator in E. coli, it allows the expression of the acrAB and tolC operons, which activate the AcrAB-TolC efflux pump, a mechanism that has been related with resistance to organic solvents, dyes, detergents, antibiotics such as chloramphenicol, tetracycline, novobiocin, erythromycin, fusidic acid and cloxacillin, as well as to cationic antimicrobial peptides, such as LL-37, HNP-2 and HBD-1.
Silent mutation (G->C) at base 132 to remove AarI and BspMI sites.
Prlux-rbs-rfp-Ter. It produces rfp when there is enough luxR and AHL.
As per BBa_K180002, omitting the RBS and STOP biobrick parts (B0034 and B0015) for speed and ease of construction.
Assess if the RBS (B0034) sequence is needed prior to GFP (E0040) whilst still able to test function of part K180000.
This promoter sequence (-80 to +1) is responsive to the transcription enhancer glnG.
An intermediate part designed to be used with the AND gate designed by Caltech iGEM '10: [http://2010.igem.org/Team:Caltech/Project Project Details].
Designed to be placed upstream of positive feedback mechanisms and desired output genes.
araC-Pbad - Arabinose inducible regulatory promoter/repressor unit
Valentina Herbring, Sebastian Palluk, Andreas Schmidt
This promoter can be induced by arabinose. In presens of arabinose the promotor is active.
Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121-30 Khlebnikov A, Skaug T, Keasling JD, modulation of gene expression from the arabinose-inducible araBAD promoter,J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.
Edited Xylose Regulated Bi-Directional Operator 2
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has a scrambled CRP binding site and and altered RNA ploymerase binding site that was designed to more resemble the consensus sequence at an intermediate level.
Both directions were kept in the sequence of the part even though it is intended to work in the right facing direction because of possible looping that may occur as a transcriptional control. The RNA polymerase binding site was altered to make binding stronger, attempting to allow activation when XylR-xylose alone is bound (the absence of CRP-cAMP).
Taken from E. Coli genome bases 3728830-3729092, base pairs changed at RNA polymerase bining sites.
Partial DLD Promoter from Kluyveromyces lactis
Tais Sineiro Herig, Raissa Estrela Curado and Gleidson Silva Teixeira
sense TWO INPUTS, activation by AHL and repression by LacI.
Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
GFP Reporter Device, Heat-Shock or IPTG Induced
Construct assembled to allow the quantitative analysis of the heat shock/LacI promoter/repressor system, BBa_K338002 . Produces GFP in response to low-temperature heat shock, more-so in the presence of LacI. GFP production is retarded in LacIq cells (or in the presence of +5mM glucose) about 4-6x. See K338002 for more detailed data.
The type of GFP used in this construct happens to have a very long half life. If growing cultures for fluorescence measurements, grow at 30??C - any higher will activate the promoter, causing GFP to be produced.
The pRE coding region from λ phage. It is able to be induced by CII (BBa_K116602).
T7 and GFP expression system due to T7 polymerase
---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Biobricking of cadC promoter and cadC gene PCR cadC-F and cadC-R on MG1655 (2336 bp, EcoR1/BamH1) Sub into pBca1256 (EcoR1/BamH1 910+2472bp, L) Product is pBca1256-K112406 ---------------------------- cb10013-cadC-F cgataGAATTCatgAGATCTcaccagcaatttgcgggcatatc cb10012-cad-R cgtagGGATCCtgaccagaccaaactggcg
The structure of toehold switch is similar to hairpin, except it has a loop at the top as ???toehold???. Toehold switch functions as riboregulator through linear-linear interaction between RNAs. When target RNA appears, it will bind one of the toehold switch stems and open the loop, exposing the RBS. Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the gene being regulated. Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 50 end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single-stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the gene of interest. The trigger RNA of our toehold is miRNA-144.When adding it to the system, GFP can be expressed.
Toehold can be open by trigger RNA, thus letting GFP express.
pCpcG2 promoter from pJT122 plasmid of Dr. Jeff Tabor. This promoter is inducible with green light through ccaR and ccaS photoreceptor system.
Search for illegal sites was performed, no sites were found.
Sequence was obtained from pJT122 plasmid of Dr. Jeff Tabor, but it was synthesized.
Promoter and ribosome binding site of the Lactococcal nisin inducible operon.
This is a ''vir'' promotor from ''Agrobacterium tumefaciens'' C58. It is located on the TI-plasmid before the ''virB'' genes. It is activated by phosphorylated ''virG'' response regulator.
PmgtCB Magnesium promoter from Salmonella
This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: BBb Standard Description Page
Paper - Primary plasmid (part 2) [tac pR, GFP and RhlI generator]
Regulated expression of RhlI (C0170) and GFP (E0040) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, E0040, B0015).
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.
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 * ''vir'' promoter from ''Agrobacterium tumefaciens'' C58, TI-plasmid
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the expresion of LasI enzyme, no terminator
The biobricks BBa_K091146 and BBa_S03154 where used to build this biobrick
Rock-paper-scissors - Rock primary plasmid
Composite part, consisting of BBa_K180011 and BBa_K180012, but with BBa_K180011 reading along reverse strand, and BBa_K180012 reading along the forward strand. Forms the 'rock' primary plasmid in Team:Southampton's rock-paper-scissors project design.
This part has been specified as a basic BioBrick, due to some of the component parts reading in reverse (unable to specify using the component BioBrick format).
Pdps promoter. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR JH089F/JH089R on Salmonella L2. (379 bp, EcoRI/BamHI/DpnI) Sub into pBjh1601CK (EcoRI/BamHI, 3134+910, L) Product is pBjh1601CK-K112322 ----------------------------------------------- JH089F Forward biobricking of {Pdps} cgataGAATTCatgAGATCTaacgattcgacgcgaaccgc JH089R Reverse biobricking of {Pdps} cgttaGGATCCaatctcatatcctcttgatg
PTAC promoter followed by an RBS, green fluorescent protein and stop codon, for testing the effectiveness of the PTAC hybrid promoter designed by iGEM2009 Team:Southampton.
Inclusion of the RBS and stop sequence to ensure correct functionality observed.
Rock - Primary plasmid (part 1) [RhlR generator]
Constitutive promoter selected to allow for continuous expression of C0171.
See relevant basic biobrick parts (J23119, B0034, C0171, B0015)
Calgary's BBa_K339007 repaired (part didn't have the sequence they said they had.
Biobricked version of Isaacs' riboregulator cis repressed lock, crR12.
Promoter of fliL gene from E. coli flagella
''fliL'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon, ). Its promoter is consitutively repressed. It is activated by flhD4C2 and by fliA with different forces ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). A deeper characterization is in project
The primers used to amplify this sequence are : Forward : TCGAATTCGCGGCCGCTTCTAGAGCAAGGGCGTGTAACAGGCAAC Reverse : TCCTGCAGCGGCCGCTACTAGTAGTCATGTGTTGCGGTCTTCCTGTG
In this portion of the design, we aim to achieve population control by combining a cell-signaling system (luxR/luxI) and suicidal gene for controlling the number of E.coli. The population controlling function in the engineered E.coli can warn us of high-density E. coli congregations by emitting fluorescent proteins. Green fluorescent light means the Cry circuit is functional and the population density is under control, while red fluorescent gene signals E.coli overpopulation.
(I) Strand C (BBa_ K332021) 1. Strand C is synthesized from three fragments ??? (1) Ptet (2) RBS+LuxR (3) RBS+LuxI+double ter sites. 2. All of them are in the form of plasmids, so we digest them by appropriate restriction enzymes. 3. We first combine the last two fragments by ligation. The procedure is as follows: (1)Add the respective digestion products, vector plasmid, buffer, ligase to ddwater (2)React at 16??C for 30 minutes (3)Inactivate the ligase by incubation it at 80??C for 20 minutes (4)Store the products at 4??C 4. The products of ligation are then transformed into competent cells. The procedure is as follows: (1)2??l plasmids(ligation products) are added to 33??l competent cells (2)Shake gently for one second, and cool it on ice (3)Incubate it in 42??C water bath for 45 seconds (4)Spread the liquid to plate which corresponding antibiotics have been added to (5)37??C overnight 5. The next day we do a colony PCR for the transformed cells and have the PCR products electrophoresis to make sure that the two fragments have been combined correctly. 6. Now we separate the plasmids(RBS+LuxR+RBS+LuxI+double ter sites) from E. Coli, and digest it with restriction enzyme. 7. The first fragment (Ptet) is then combined with the above product by ligation, and transformed into competent cells by the same we have done early. 8. Because the length of Ptet to the remain segment is relatively too short, we cannot confirm whether they have combined correctly by just do an electrophoresis of the colony PCR products. If the electrophoresis result seems like that they have combined, we then send the product to sequencing.
1. LuxI protein (BBa_C0061) is the synthase that converts S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). When the concentration of AHL reaches a threshold level, it binds to the N-terminal domain of a LuxR protein. Then the LuxR/AHL complex binds the Lux box within PtetR promoter (BBa_C0040), which then activates suicidal gene expression. 2. LuxR (BBa_C0062) is transcribed continuously by the constitutive promoter (BBa_J23106), and later works in conjunction with AHL. This design can control the concentration of LuxR protein in a steady phase, and the concentration of AHL/LuxR complex is only affected by the concentration of LuxI protein (BBa_C0061).
The part codes for the human Oct-4 promoter, which regulates expression of the Oct-4 gene. The Oct-4 gene is essential for pluripotency in cells and is downregulated once differentiation occurs. Sequence information is from GenBank (NM_002701) and shows the cis-regulatory mRNA that controls transcriptional activation of the human Oct-4 promoter.
Raissa Estrela Curado, Tais Sineiro Herig and Gleidson Silva Teixeira
position 3 mutated promoter lux pR-3 (luxR & HSL regulated)
The position 3 mutated promoter lux pR-3 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 3 mutated promoter lux pR-3 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-3 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
In the promoter lux pR's sequence, the C residue of position 3 was changed to a T using PCR method.
The position 3 mutated promoter lux pR-3 is based on promoter lux pR (BBa_R0062).
V.cholerae ctx gene promoter region. Transcription takes place In the presence of a ToxR dimer. We don't have the -10 and -35 sites annotated (need to do this).
GENBANK M21250
K091146 is a modified version of the pLas' promoter (BBa_K091117) containing the addition of a luxR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of PAI-1 and LasR and repressed in the presence of AI-1 and LuxR.
The Lux box was placed adjacent to the LasR binding site with the aim of minimizing cross-talk in the presence of both PAI-1 and AI-1; since the LasR site should have a higher affinity, we hope that the binding of PAI-1 will competitively exclude the binding of AI-1.
The LuxR binding site is from BBa_R0062 and the pLas' promoter is from the region upstream of the LasI gene in Pseudomonas aeruginosa.
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">
Gram-Positive Strong Constitutive Promoter
This is a strong constitutive (always-on) promoter for use with gram-positive organisms. It requires a downstream gram-positive ribosomal binding site and coding sequence to function. We only have experience using this part in B. subtilis, but it should function in most gram-positive organisms. It has not been tested and will most likely not work in E. coli. The promoter comes from the cis-regulatory region of the uracil phosphatase in Bacillus cereus. We functionally annotated the promoter ourselves using the DBTBS database motif search feature. (dbtbs.hgc.jp)
This part generates high expression in gram-positive organisms, and it may be possible to generate deleterious effects on the cell if used to express certain protein products.
We PCRed this part out of the pAD43-25 gram positive vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE166.
Rock-paper-scissors - Rock secondary plasmid [rhl pR, LacI generator]
iGEM2009 Team:Southampton Game of Life secondary plasmid.
Regulated expression of LacI (C0012) by Rhl pR (R0071).
See relevant basic biobrick parts (R0071, B0034, C0012, B0015)
VirA/G signaling system without read-out
This BioBrick is a virA/G signaling system without read-out. It contains an unmutated ''virA'' gene and a ''virG'' gene that works without ''rpoA'' from ''A. tumefaciens'' and a ''vir'' promoter.
This BioBrick can be used to control any gene with a ''vir'' promoter
''A. tumefaciens'' C58 TI-plasmid, partsregistry, Mr. Gene
Promoter of flgA gene from E. coli flagella
''fliA'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCATATCTCCTCCGCAGGTATCAAAAT *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAACAGTATCGCGATGATCGCCACGCTACGT
The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the expression of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. This composite part does not have an LVA degradation tag at its end, hence can be used with all prokaryotic assemblies.
The part was designed and assembled from the iGEM2009 kit with standard RFC10 assembly.
The AraC promoter is found naturally in E.Coli at 1.52 min/centisome in the clockwise orientation. More information can be found at [http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene].
This is the promoter for the ''Eschaerichia coli'' JM109 ''cstA'' gene. It includes the CRP-binding site and the RNA polymerase-binding site. Low glucose concentration results in increased activity by adenylate cyclase. cAMP binds to the cAMP receptor protein, which, in its bound form, is able to associate with the promoter and promote transcription of the downstream gene. (''cstA'' encodes the carbon starvation protein.)
This biobrick has a promoter inducible by PAI+LasR (BBa_R0079)and produces LuxI enzyme (BBa_F1610). This enzyme produces 3OC6HSL (AI).
The DNA of BBa_R0079 and BBa_F1610 biobricks where used.
Promoter for grpE gene - Heat Shock and Ultrasound Sensitive
According to Vollmer, et. al in their 1998 Applied and Enviormental Microbiology paper, "Bacterial Stress Responses to 1-Megahertz Pulsed Ultrasound in the Presence of Microbubble", the promoter for the grpE gene, a known heat shock promoter, is upregulated 24 times when cells are exposed to high frequency ultrasound and microbubble cavitation.
Three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5′-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43.
We designed the sequence of bc3-5 by add perfix and suffix which are belong to BioBrick RFC 10.
From Bacillus thuringiensis subsp. chinensis CT-43 In the CT-43 genome19,20 (GenBank accession: NC_017208.1), five type I c-di-GMP riboswitches named Bc1, Bc2, Bc3, Bc4 and Bc5 RNA (Supplementary Tables S1???S5) were annotated in the Rfam database (http://rfam.sanger.ac.uk/search). Zhou, H., et al. (2016). "Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter." Sci Rep 6: 20871.
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
P2 promoter in agr operon from S. aureus
The agr P2 operon is an autocatalytic sensory transduction system in Staphylococcus aureus. P2 promoter regulates the synthesis of AgrB (a transmembrane protein) and AgrD (precursor of AIP autoinducing peptide). AIP binds to AgrC, a membrane binding receptor and it phosphorylates AgrA. The phosphorylated AgrA increases the expression level from P2 promoter.
Codon usage has been optimised for E. coli (which also seems to give good results for B. subtilis
The section of the sequence constituting the P2 promoter is subject to rather more debate than the sequences of the coding regions. [http://jb.asm.org/cgi/content/full/186/22/7549 Koenig, Robbin L., Ray, Jessica L., Maleki, Soheila J., Smeltzer, Mark S., Hurlburt, Barry K. Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region J. Bacteriol. 2004 186: 7549-7555] shows the areas of the sequence to which phosphorylated AgrA binds while L Rao, R K Karls, and M J Betley: In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus. Bacteriol. 1995 May; 177(10): 2609???2614. shows the transcription start sites; we took the overall extent of the promoter region from that labelled in Morfeldt, E., K. Tegmark, and S. Arvidson. 1996. Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus. Mol. Microbiol. 21:1227???1237. (not available online).
This is the PLL promoter taken from the P4 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PLL promoter.
Promoter of the FIG1 gene in yeast. The transcription factor Ste12, activated in the yeast pheromone pathway, binds to the FIG1 promoter and initiate the expression of the FIG1 gene. Five pheromone response elements (PRE, consensus sequence ATGAAACA) have been identifi?ed in the 5'-upstream regulatory sequence of the FIG1 gene. Phosphorylated Ste12 binds to them and thereby activates transcription. The PREs in the FIG1 promoter di?ffer slightly from the PRE consensus sequence ATGAAACA: Sequence Location TGAAACG -405 TGAAACC* -217 TGACACA* -206 TGAAACG -173 TGAAATA -133 Sites marked with an asterisk occur on the opposite strand The strictly regulated transcriptional feature of the FIG1 promoter can be useful when investigating the pheromone pathway. The promoter can be used to express reporter genes in response to activation of the pheromone pathway. Reference: Erdman S, Lin L, Malczynski M, Snyder M. Pheromone-regulated Genes Required for Yeast Mating Diff?erentiation. The Journal of cell biology. 1998;140(3):461-483.
The length of the promoter was chosen to 450 bp as recommended by Kondo group of Kobe University of Japan. Togawa S, Ishii J, Ishikura A, Tanaka T, Ogino C, Kondo A. Importance of asparagine residues at positions 13 and 26 on the amino-terminal domain of human somatostatin receptor subtype-5 in signalling. J Biochem. 2010 Jun;147(6):867-73
The sequence has been amplified directly from genomic yeast DNA.
Tadashi Nakamura, Shuhei Yasumoto, Takahiro Saka, Saya Kakuda
LacI promoter with RBS attached downstream.
Registry parts BBa_R0010 (promoter) and BBa_B0034 (RBS).
K091157 is a modified version of the pLux' promoter (BBa_K091156) containing the addition of a LasR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of AI-1 and LuxR and repressed in the presence of PAI-1 and LasR.
In order to fit the LasR binding site between the -35 and -10 regions, the last base of the Las Box was cropped and the -35 region was modified to overlap with the first two bases of the Las Box.
The LasR binding site is from the region upstream of the LasI gene in Pseudomonas aeruginosa. The rest of the promoter is the same as BBa_K091156.
promoter of phoB in E.coli, an external phosphate regulated promoter which can be activated when encountering phosphate limitation.
The promoter region should contain PhoB binding site
This is suggested to be an adhesion responsive promoter. It contains the binding site for CpxR-P (phosphorylated CpxR). CpxR is one member of the two-component Cpx response to envelope stress. Briefly, envelope stress causes CpxA, an inner-membrane histidine Kinase to Phosphorylate CpxR, allowing it to bind to CpxR-P responsive regions and regulate downstream target genes. See 'Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway', Karen Otto and Thomas J. Silhavy, PNAS, Vol. 99., No, 4., 2287-2292. 2002.
This part comes from the genome of E.coli TR235, as used in the PNAS paper of Otto and Silhavy mentioned previously. TR235 is K-12 MC4100 containing a pCpxR-LacZ fusion inserted at the (Lambda)att site, plus some other modifications (see Otto and Silhavy). The part is a portion of the CpxR responsive region used in these LacZ fusions.
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 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.In thisversion, a binding site for cI dimer has been added: binding to this site dominantly represses transcription.
V. fishceri and phage lambda (courtesy of Ron Weiss)
PCR mv028/mv029 on rrnB native sequence (721bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112118 ----------------------------------------------- mv028 Forward Biobricking of rrnB P1 promoter cgataGAATTCatgAGATCTatgcactaaaacgtatcctacgcccg mv029 Reverse Biobricking of rrnB P1 promoter cgttaGGATCCcgccttcccgctacagagtcaagc
Promoter of xpsR (PxpsR) that responds to the Quorum Sensing signal 3 OH-PAME especific of Ralstonia solanacearum.
mCYC promoter plus Zif268-HIV binding sites
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the Zif268-HIV binding domain. It contains the sequences of parts BBa_K165011 and BBa_K165016 but also contains additional DNA.
It was necessary to design primers for use in PCR to remove this part from its host plasmid and transfer it to a standard Biobrick vector.
Composite part that was taken via PCR from a synthetic plasmid containing the sequence. The plasmid came from Lawrence Berkeley National Laboratory.
I0500 with extra trailing restriction sites: Eco-Xba-I0500-HindIII-NcoI-Spe-Pst
This promoter was isolated by taking 500bp upstream of the MF(ALPHA)1 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
This gene is on the Watson strand of the entered sequence.
Rick Carrick, Ambhi Ganesan, Alex McMillan
This is the same sequence as MFA1 promotor(BBa_K110008), but with suffix and prefix switched
K338001+R0011: Heat Shock Promoter + LacI Regulated Promoter
Fused heat shock promoter with lacI regulated promoter. It possesses the characteristic of both heat shock promoter and LacI.
It was designed to address the issue of a leaky promoter in front of a bacterial gene in an effort to create a genetic switch just for growth/cloning
Modified lambda P(RM) promoter: -10 region from P(L) and cooperatively repressed by 434 cI
Lamdba P(RM) promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operator sites are used for both activation and repression to enhance cooperativity. Additionally, the -10 region from lambda P(L) was used instead of that from P(RM).
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage. This part differs from BBa_I12036 because the -10 region from P(L) was used instead of that from P(RM).
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40. ; Lutz, R & Bujard, H. "Independent and tight regulation of transcriptional units in Escherichia coli via the lacR/O, the TetR/O and AraC/I1-I2 regulatory elements, Nucleic Acids Research, 1997, Vol.25, No.6 (1203-1210)
Modified lamdba Prm promoter (cooperative repression by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operators are used for both activation and repression to enhance cooperativity.
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage.
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40.
An 328 bp promoter region, which can be assembled with urea binding proteins to activate the downstream gene expression.
pUreD is a urea responsive element that should be turned on by the complex of UreR and urea.
This is a component of urea sensor cloned from Proteus mirabilis.
The sicA promoter is a regulatory element in the Salmonella SPI-1 secretion system. It controls the expression of secreted effector proteins in the natural system and turns on late. Please reference Temme et al, 2008, JMB for exact dynamics of this promoter and associated feedback mechanisms. This promoter only functions in Salmonella strains containing SPI-1.
This part can only be used in salmonella strains.
5' upstream sequence of qrr4 containing -12 and -24 regions (specific for Sigma factor 54)
>gi|156527546:c1833759-1833376 Vibrio harveyi ATCC BAA-1116 chromosome II, complete sequence
promoter to xylF without CRP and several binding sites for xylR
removed RBS crp binding and xylR binding sites
The promoter PAtpI has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 min of germination [https://www.ncbi.nlm.nih.gov/pubmed/25661487 Sinai''et al.'']. This gene is part of an operon, therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. The region was checked for the binding of sigma factors and transcription factors with DBTBS. No binding factors were found with the highest significance level. In our project we wanted to find a constitutive ON promoter for our ciprofloxacin resistance casette. In the following part we put the promoter PAtpI in the pSB1C3 backbone to make it available to other iGEM teams.
Ordered as gBlock. IDT (Integrated DNA technologies).
Zif268-HIV binding sites + TEF constitutive yeast promoter
Complex Quorum sensing circuit that receives the signal of PAI+LasR and AI+LuxR to control the production of LuxI and LasI enzymes.
The biobricks BBa_K266006 and BBa_K266000 where used to build this biobrick
The Pseydomonas quinolone signal (PQS) as well as its precursor 2-heptyl-4-quinolone (HHQ)are very important in mechanisms controlling virulence in Pseudomonas aeruginosa. P. aeruginosa has a key role in Cystic Fibrosis (CF). PQS can initiate signalling for various processes like virulence, motility, and biofilm formation. A good number of these processes are facilitated by PqsR, which is a transcriptional factor. An activated PqsR will go to the nucleus of the cells and activate the respective genes. http://www.ncbi.nlm.nih.gov/pubmed/21539583 http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs http://www.ncbi.nlm.nih.gov/pubmed/12393198
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector.
The source of this part is the genome of Pseudomonas aeruginosa PAO1 provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Composite part, consisting of BBa_K180005 and BBa_K180006, but with BBa_K180005 reading along reverse strand, and BBa_K180006 reading along the forward strand.
BBa_K180005 reading along reverse strand, and BBa_K180006 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180005) has no effect over LacI or GFP-LVA expression in BBa_K180006.
See relevant biobrick component parts (BBa_K180005 and BBa_K180006)
Positively regulated, OmpR-controlled promoter. This promoter is derived from the upstream region of ompC. Phosphorylated OmpR binds to the operator site and activates transcription. This is a truncated version of BBa_R0082.
The promoter deletes the C2 and C3 regions of BBa_R0082 (bases 37-74 in BBa_R0082) and inserts an 8-base linker before the -35 and -10 signal region. The efficacy of this promoter versus the full ompC upstream region (BBa_R0082) or the ompF upstream region (BBa_R0084) is currently unknown. The a at base 41 is mutated to t to avoid XbaI site. Speculation that this was a previous assembly site.
Construct assembled to allow the quantitative analysis of the heat shock promoter, K338001 . Produces GFP in response to low-temperature heat shock.
The type of GFP used in this construct happens to have a very long half life. If growing cultures for fluorescence measurements, grow at 30°C - any higher will activate the promoter, causing GFP to be produced.
Double Promoter (LuxR/HSL, positive / cI, negative)
Double Promoter (LuxR/HSL, positive / P22 cII, negative)
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. This part also exist together with lacI, part BBa_K180000 [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Oligos ordered from SigmaAldrich were annealed to form a double stranded DNA segment, ready to be ligated into any BioBrick backbone.
Created from oligos ordered from SigmaAldrich. Oligo sequences: Ptac+ AATTCGCGGCCGCTTCTAGAGGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTA Ptac- CTAGTAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACAGCTCCTCTAGAAGCGGCCGCG
nhaA promoter, that can be regulated by pH and nhaR protein.
It's E.coli K12 sodium/proton anti-transport promoter. It can be a pH sensor. We test pH senses in different pH value and Sodium concentration.
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes the three OmpR binding sites: C1, C2, and C3. The promoter starts at -107 and goes up to the transcriptional start site at +1. An alternate version, BBa_R0083, cuts out the C2 and C3 sites. The efficacy of this promoter versus the truncated ompC upstream region (BBa_R0083) or the ompF upstream region (BBa_R0084) is currently unknown.
This is the Psid promoter taken from the P4 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the Psid promoter.
MC7-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter. It is a test of our positive feedback circuit for microcystin.
MC7, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
Scissors - Primary plasmid (part 1) [LasR generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'scissors' primary plasmid.
Constitutive promoter selected to allow for continuous expression of C0179.
See relevant basic biobrick parts (J23119, B0034, C0179, B0015)
PETase gene encodes the PETase enzyme which can degrade PET to terephthalic acid (TPA) and ethylene glycol (EG) with another enzyme MHETase. However, the product TPA can cause inhibition of the reaction and slow down it. This expression system consists of the TPA inducible Leader sequence, the enhanced promoter PGK1(BBa_K2110012), the PETase gene(BBa_K2110800) and Terminator CYC1(BBa_K2110000). It can be induced by product TPA so that this is a positive feedback system. This system is used in Saccharomyces cerevisiae.
Edited Xylose Regulated Bi-Directional Operator 1
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has a scrambled CRP binding site and and altered RNA ploymerase binding site that was designed to more resemble the consensus sequence slightly.
Hybrid promoter: HSL-LuxR activated, P22 C2 repressed
Hybrid promoter consisting of the Lux box which overlaps partly with -35 box. Binding sites for P22 C2 OR2 and OR1 are located between the -35 and -10 boxes and downstream of them respectively.
-35 box is partly composite. -10 is taken from the P22 phage PR regulatory region
This promoter was isolated by taking 500bp upstream of the STE3 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
Our microarray experiment showed that this gene was responsive to sound and ultrasound. Therefore, we cloned the promoter region located approximately 250 base pairs before the start codon for the cadB gene.
There was a relatively high basal level of expression of this gene that made this part difficult to incorporate into our lysis device design.
cloned from E. coli MG1655 genome located upstream of CadA and CadB genes
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the LexA binding domain.
Composite part was taken via PCR from a synthetic plasmid containing the sequence. The plasmid came from Lawrence Berkeley National Laboratory.
Right facing promoter (for xylF) controlled by xylR and CRP-cAMP
potential looping feature possibly removed
removed RBS, left promoter, and several xylR binding sites
Encodes a transcription activator for the right handed Lux promotor. Two molecules of acyl-homoserine lactones (AHL) form a complex with two molecules of the LuxR protein. This complex then binds to a palyndromic site on the lux promoter, increasing its transcription rate. LuxI, LuxR and the right lux promoter are all derived from V. fischeri.
Biobrick BBa_C0061 still contained a Barcode sequence which was deleted. For our own use, 9 bp were exchanged to create unique restriction sites and to optimize DNA folding.
Sequence is originating from V. fischeri. Here, the sequence from BBa_I0462 was used and optimized.
The pLux' promoter is a modified version of BBa_R0062 containing the -35 region TTTAAT in place of TTTACG. Transcriptional activation is expected to occur in the presence of LuxR and AI-1, just as with R0062. The modified -35 region was designed to serve as a control for the evaluation of BBa_K091156, the pLux+ Las- promoter.
This promoter is identical to pLux+ Las- with the exception of the Las box's absence
The manA promoter from Xanthomonas campestris
The source of this part is Xanthomonas campestris pv. campestris 8004 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Autoinducer synthetase for acyl-homoserine lactones (AHL): LuxI synthesizes AHL (3OC6HSL). Two molecules of AHL form a complex with two molecules of the LuxR protein. This complex then binds to a palyndromic site on the lux promoter, increasing its transcription rate. LuxI, LuxR and the right lux promoter are all derived from V. fischeri. In absence of AHL, the right Lux promotor only gives weak constitutive expression of downstream genes. Any gene you would like to be controlled by AHL and thus cell density can be cloned after this complex.
For the ease of cloning, the sequence was devided in multiple gBlocks. Therefor, there is a mistake in the composite part sequence since the scar is only partly present.
The Lux operon originates from V. Fischeri.
RHLR/RHLI Inducible & LasR/LasI repressible Promoter
This part is similar to BBa_R0079 but has an added binding site for RHLR/RHLI in between the -35 and -10 region. Thus this promoter is induced by LasR/LasI but repressed by RHLR/RHLi binding.
There is a 17bp spacer between the -35 and -10 regions and the binding site for RHLR/RHLI is 20bp long. I was able to fit the whole binding site in the spacer region because the last three base pairs of the -35 region and the first three base pairs of the RHLR/RHLI binding site are the same.
Rust L. et. al., "Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Journal of Bacteriology, 1996.
CinR, CinL and glucose controlled promotor
This promotor is controlled by cinr, cinl and extrcellular glucose concentration. High extracellular glucose concentration will inhibit this promotor through the action of phosphorylated OmpR. Phosphorylated OmpR will bind the OmpR binding site of the promotor and block the movement of RNA polymerase. In contrast, the combined action of CinR and CinL will turn on the promotor. But only when the extracellular glucose level is low so OmpR dephosphorylate and dissociate from its binding site will this promotor become active. In essence, this promotor function as an AND gate, it will turn on if and only if the extracellular glucose level is low and there are some functional CinR and CinL protein inside the cell.
The key to the function of the promotor is the inhibitory ability of OmpR binding site. To ensure maximum affinity with phosphorylated OmpR, we use all three OmpR binding site.s.
This hybrid promoter is designed to respond to activation by LuxR when it is bound to AHL and to repression by the phage lambda cI repressor. The repression needs to win over activation.
This part is based on the idea of replacing the region of the Lux promoter downstream of the -35 with the OR2 cI half-operator, added LuxR-10 region. Based on what we have found up to now, promoters which are constructed just by inserting OR1/2 downstream of Lux are all very leaky.
this part comes from LuxR promoter and cI lambda promoter.
Promoter, Standard (luxR and HSL regulated -- lux pR)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr Drew Endy
[Note: This is the same part as R0062 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. 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 part=BBa_R0062>Image1.gif</bb_file>" width="614" height="362"> Modified to comply with BBa_S0001: TTGACA-17N-GATACT
Lead Promoter (Part 4) messed up EcoRI site
Promoter of the copA copper efflux gene from Escherichia coli K12. This promoter is induced in the presence of copper ions, regulated by the activator protein CueR (CopR).
Promoter of flgB gene from E. coli flagella
''flgB'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACAGTATCGCGATGATCGCCACGCTACG *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGCATATCTCCTCCGCAGGTATCAAAATT
This is the GAL1 inducible promoter for use in Saccharomyces cerevisiae. It is repressed in the presence of glucose and induced in the presence of galactose. Raffinose is a neutral sugar source the neither induces nor represses the promoter. pGAL1 normally has an "all-or-nothing" response to galactose, but Christina Smolke has shown that knockout of the Gal2 gene gives pGAL1 a tunable, linear response to galactose.
From the Saccharomyces cerevisiae genome, via Caroline Ajo-Franklin.
This is the PP promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PP promoter.
pVeg SpoVG T7 SpoVG RFP double terminator.
T7 and RFP expression system in B. subtilis
This part consists of the enhanced promoter PGK1(BBa_K2110012), the tpaR gene(BBa_K2110002), and the terminator CYC1（BBa_K2110000）. The tpaR gene encodes the terephthalic acid (TPA) positive feedback regulatory protein. This protein can bind the TPA inducible leader sequence (TILS) at the existence of TPA and induce the expression of downstream gene. The tpaR protein belongs to the IclR protein family and has helix-turn-helix domain.
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of our target gene gp35 through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, which alone can set a DNA sequence(there it means the promoter) flanked by oppositional attB and attP sites, thereby produce an inverted sequence flanked by attL and attR sites. As a consequence, the promotor moves on to express the toxic protein instead of the attractant. We insert the gp35 gene into the downstream region of the PompR promotor by means of 3A synthesis so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation. Meanwhile, we insert the recombined sites of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we accomplish our reverse promotor system.
we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation.
Zif268-HIV binding sites + MET25 constitutive yeast promoter
pu promoter drives transcription of the upper operon of Pseudomonas putida mt-2 TOL plasmid for degradation of toluene and xylenes. pu is activated by the activator protein XylR (BBa_K1946005) in the presence of toluene and xylenes.
Synthetically synthesized from GeneWiz based on Pseudomonas putida TOL plasmid
Binding region for LasR protein (positive regulation)
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
LasR/LasI Inducible & RHLR/RHLI repressible Promoter
The natural spacer between the -35 and -10 is 14bp and the natural O1 site for RHLR/RHLI is 20bp long. There was not much over lap of the -35 or the -10 with this sequence, so it was nessicary to chop off some of the binding site sequence in order to maintain the correct spacing and preserve the -35 and -10 sequence. The binding site sequence is missing a tcc on is 5' end and a t on its 3' end.
unidentified/uncharacterized CinR dependent
TPA transporting protein gene expression system
This part consists of the enhanced promoter PGK1(BBa_K2110012), the tpaK gene(BBa_K2110003), and the terminator CYC1（BBa_K2110000）. The tpaK gene encodes the terephthalic acid (TPA) transporting protein. TPA is not a kind of nutrient substance for most organisms, including common chassis organisms like E.coli and Saccharomyces cerevisiae. However, as a kind of TPAdegradable organism, RhodococcusjostiiRHA1 can transport TPA into their cells because of the TPA transporting protein gene tpaK in their genome. This gene is also verified to be useful in Saccharomyces cerevisiae.
The pqsA promoter from Pseudomonas
The Pseudomonas quinolone signal (PQS) is imperative in modulating mechanisms that enable Pseudomonas aeruginosa to survive at times of stress. P. aeruginosa is the main pathogen responsible for severe deterioration in the Cystic Fibrosis lungs. The production of this signal relies on the pqsABCDE operon. The transcriptional factor PqsR (BBa_K1315000) positively regulates this operon. http://www.ncbi.nlm.nih.gov/pubmed/25225275 http://www.ncbi.nlm.nih.gov/pubmed/16735731 http://www.ncbi.nlm.nih.gov/pubmed/21539583 http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs http://www.ncbi.nlm.nih.gov/pubmed/12393198
LasR and 3OC12HSL regulated promoter with CI Operator 1 (binding site for lambda cI repressor)
Hybrid promoter (trp & lac regulated -- tac pR)
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. The Ptac sequence has been extracted by PCR from the commercially available plasmid pMAL-pIII from New England Biolabs (N8101S), with the addition of the biobrick prefix and suffix sequences. [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Sequence includes lacIq and Shine-Dalgarno sequence from pMAL-pIII prior to the Ptac promoter to ensure correct functionality of the promoter.
Scissors - Primary plasmid (part 2) [tac pR, mBanana and LuxI generator]
Regulated expression of LasI (C0178) and mBanana (K180009) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180009, B0015).
LsrA promoter (indirectly activated by AI-2)
The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Once uptaken into the cell, AI-2 will be phosphorylated under the effect of LsrK. Without the presence of AI-2, LsrA promoter is inhibited by the repressor, LsrR. Phosphorylated AI-2 will prevent LsrR from inhibiting this promoter, hence activating it so that the downstream sequence can be expressed. To prevent the cell from self-inducing this promoter, we must suppress its AI-2 producing capability. It can be done by knocking out LuxS, one of the vital gene required for producing AI-2. By doing that, LsrA promoter can only be activated under the presence of AI-2 from other sources than the host cell which is LuxS mutant (say, by contact with normal cells). More information on AI-2 quorum-sensing (QS) system please refer to: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1952038
This is the same sequence as STE2 promotor(BBa_K110008), but with suffix and prefix switched
A part used to quantify the AraC-RBS construct K259007 created by the BCCS-Bristol iGEM'09 team.
This part was designed with the specified biobricks from the parts registry. AraC occurs naturally in E.coli. GFP occurs in jellyfish species, RBS designed by the Anderson family and Terminator by Resma Shetty.
This a GFP reporter regulated by an allosteric intron responsive to theophylline.
The ligand response is alter from c-di-GMP to theophylline by merging a theophylline hammerhead ribozyme to the intron.
The intron derives from the genome of Cd
The clp gene from Xanthomonas campestris
c-di-GMP Repressed DNA Binding Protein (Clp) Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads. http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris
The sequence had 1 PstI site that was mutated out by site-directed mutagenesis.
The promoter region (inclusive of regulator binding sites) of the B. subtilis RocDEF operon
This is the reported promoter region from the Bacillus subtilis RocDEF operon as taken from the DBTBS website (http://dbtbs.hgc.jp/). Base 1 to base 21 contains the binding site for the binding factor, RocR. The sequence, TATGCAAAAGAATTTTGCACT, reportedly contains the consensus sequence for binding of this factor. Base 42 to base 62 contains another binding site for the RocR binding factor, with ATATCAGAATGTTTTTGCACC as the binding consensus for this sequence. Bases 113-135 outline the binding site of the binding factor AhrC (CTTGCATTTATATAAAGGGAAAG). Bases 96 to 135 outline the promoter region for this operon (CTTGATTTGGCACAGAACTTGCATTTATATAAAGGGAAAG).
This sequence only outlines the reported promoter region as ascertained from the online resource described above, and may lack nucleotides not described by the operon analysis used.
Information was obtained from: http://dbtbs.hgc.jp/COG/prom/rocDEF.html.
Natural Xylose Regulated Bi-Directional Operator
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has an unscrambled CRP binding site and and unaltered promoter region (unlike BBa_K100001 through BBa_K100003).
Both directions were kept in the sequence of the part even though it is intended to work in the right facing direction because of possible looping that may occur as a transcriptional control.
Taken from E. Coli genome bases 3728830-3729092
position 5 mutated promoter lux pR-5 (luxR & HSL regulated)
The position 5 mutated promoter lux pR-5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-5 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-5 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
By PCR method the G residue of position 5 was changed to a C based on the sequence of the promoter lux pR(BBa_R0062).
Game of Life - Primary plasmid (part 2) [lux pR, GFP and LacI generator]
Regulated expression of LacI (C0012) and GFP (E0040) by PLUX promoter (R0062), controlled by presence/absence of AHL-LuxR.
See relevant basic biobrick parts (R0062, C0012, B0034, K180001, B0015)
THIS PART DOES NOT WORK. Need to add the CI operator sites.
This is the PO promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PO promoter.
The source of the DNA is the P2 phage genome.
Modified lamdba Prm promoter (repressed by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI)
The O-R1 region of 434 contained 14 base pairs as opposed to the 17 base pairs of the O-R3 site of lambda. Also, it was noticed that the O-R3 site of the lambda included part of the -10 site. Hence, to preserve the spacing and the -10 site, the three nucleotides that were in both the -10 site and the lambda O-R3 site were retained. The 14 nucleotides that were in the O-R3 site and not in the -10 site were replaced with the O-R1 site of the 434.
Promoter driving the lasI gene in Pseudomonas aeruginosa. Activated by LasR+3OC12HSL and not RhlR+C4HSL (Seed et al., 1995) ~4x stronger than the lasB promoter (BBa_R0079; Seed et al., 1995). Switch point ~0.1nM 3OC12HSL (Seed et al., 1995)
Promoter sequence cropped to 4nt upstream of Las box according to example of Whiteley and Greenberg, J. Bac, 2001
Binds Maltose and stimulates transcription
Weaker version of wild type pBAD ( I13453 ).
Site-directed mutagenesis on I13453 with the following primers: Forward: TAATCTTATGGATAAAAAAGCTATGGCATAGC Reverse: GCGGATCCTACCTGACGCTTTTTATC
Promoter sequence with recognition site for CueR transcriptional regulator protein.
this hybrid promoter is designed to be consecutively off.It combines with plasR(R0079) and lambda cI OR1(see C0051).Adding P-AI-1 sould activate the promoter, while repressed by lambda cI.
"CI binding of the OR region. It binds OR1 with highest affinity, OR2 with next highest, and OR3 with lowest"(http://www.nonanone.com/qw/files/Biology%204450/test2_lecture8,9.txt)
MC31-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter It is a test of our positive feedback circuit for microcystin.
MC31, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
Pmom is one of the four late promoters of the Mu bacteriophage. It can be activated by the C protein, which is produced by the middle promoter Pm. Pmom works because of a special configuration of the DNA. It can be used to create a sensitivity tuner alongside the C protein of the same phage. This construction can be used to increase the POPS output of a promoter.
The design of this part had no special considerations
Synthetized from the reported sequence of Pmom promoter from Mu bacteriophage
This part has the arabinose regulated promoter (AraC/pBAD) upstream of a ribosome binding site. This is ideal for inserting a protein to be expressed downstream of this part as it's expression can be regulated by the presence of the concentration of arabinose in the growing environment/media of the cells. ==What this part actually does== This composite part includes the AraC/pBAD promoter and a strong ribosome binding site designed from the Anderson family of RBS. The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the trancription of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. Controlling the transcription will not ensure translation. The ribosome binding site is ideally placed a small distance away from the promoter site to ensure that the ribosome will bind and start translating the protein coding sequence found downstream.
The part was designed with RFC10 assembly from supplied biobricks of the iGEM2009 distribution.
The AraC promoter can be found naturally at 1.52min of the E.coli genome ([http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene number EG10054]. The RBS was taken from the designed sequences of RBS by the Anderson family. For more information visit the original part page [http://partsregistry.org/Part:BBa_J61100 here].
Rock-paper-scissors - Paper secondary plasmid [lux pR, LacI generator]
iGEM2009 Team:Southampton rock-paper-scissors 'Rock' secondary plasmid.
Regulated expression of LacI (C0012) by Lux pR (R0062).
See relevant basic biobrick parts (R0062, B0034, C0012, B0015).
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type ( I13453 ) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
Game of Life - Secondary plasmid [tac pR, LuxI generator]
Regulated expression of LuxI (C0161) by TAC pR (K180000), a functional hybrid lac/trp promoter.
See relevant basic biobrick parts (K180000, B0034, C0161, B0015).
Phns in BBb1 format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of [<Phns>] Basic Part K112701 PCR dv001/dv006 on Phns from E. coli K12 MG1655 (~342 bp, gp = A) PCR dv005/dv003 on Phns from E. coli K12 MG1655 (225 bp, gp = B) PCR dv004/dv002 on Phns from E. coli K12 MG1655 (~149 bp, gp = C) --------------------------------------------------- PCR dv001/dv003 on A+B (355 bp, gp = D) --------------------------------------------------- PCR dv001/dv002 on C+D (697bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+697) Product is pBca1256-K112701 ----------------------------------------------- dv001 Forward Biobricking of [<Phns>] CgATAgaattcATGagatctGAAATATAGCTGTGCCATCAGCC dv002 Reverse Biobricking of [<Phns>] cagtcggatccGCACGAAGAGTACGGATG dv003 Removing the 2nd EcoRI site in [<Phns>] (R) GCCAGGAATGTAAGGgATTCAAAATTGTTC dv004 Removing the 2ND EcoRI site in [<Phns>] (F) GAACAATTTTGAATcCCTTACATTCCTGGC dv005 Removing the FIRST EcoRI site in [<Phns>] (F)CGCTTAATAGGGgATTCTCGTAAACAC dv006 Removing the FIRST EcoRI site in [<Phns>] (R)GTGTTTACGAGAATcCCCTATTAAGCG
Based on the AHL regulated BioBrick part R0062 and the previous work on LacI regulated promoter (ref. 1), we designed a new promoter regulated by AHL and LacI. Since this promoter is a kind of a "HYBRID" of the two, we call it HYBRID PROMOTER. Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
Promoter activated by RhlR in concert with C4-HSL. C4-HSL is produced by RhlI, and acts as a quorum-sensing autoinducer. This is the natural sequence taken from Pseudomonas aeruginosa. Crosstalk: this promoter is also activated at a low level by LasR with its associated HSL.
The -10 and -35 sites are labeled as identified in [Pearson97]. The part begins with the RhlR binding site, and (rather arbitrarily) ends with the first transcribed base (+1).
Pseudomonas aeruginosa rhlAB promoter
This is a modification of the standard plac operon with everything before the -35 site removed, the Or2 site from the lambda promoter inserted at the beginning, and a 17-bp spacer added between the two.
This pLas' promoter contains the regulatory region upstream of the LasI gene in Pseudomonas aeruginosa. K091117 includes a LasR binding site as well as a -10 region modified from CATAAA to TATAAA to reflect our pLasXOR promoter design. Activation is expected to occur in the presence of the autoinducer PAI-1 and the LasR protein. The pLas' promoter serves as a control for the evaluation of the pLasXOR promoter, part BBa_K091118.
To minimize leaky transcription, a secondary transcriptional start site was excluded from the design.
The sequence of the LasI upstream regulatory region is available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=176640
AraC operator, truncated to include araO1, araI1, araI2, c-amp1, and c-amp2 sites. This operator should *activate* transcription in the presence of AraC; b/c the operator lacks the araO2 site, there should not be araC-mediated repression.
Transciptional regulator reliant on screams of someone, somewhere in trouble.
DNA sample - Taken from Clark Kent's Coffee Mug at the Daily Planet
nir promoter from Synechocystis sp. PCC6803
The ''nir'' promoter is nitrate inducible.
The ''nirA'' promoter originates from ''Synechocystis'' sp. PCC6803.