Built by Josh as an intermediate in screening plasmid construction.
This part is an intermediate to the protein generator BBa_45300. This part is almost identical to BBa_J45299 except that is uses as its RBS B0032 instead of B0030.
Thermo sensitive small RNA (ROSE structure) which can be used as a RNA regulator.
Part of the sequence is altered because of the scar...
predicted stem-loop extending to one position before start codon
This part accepts POPS to generate EYFP under the medium strength RBS B0032.
This is the venus YFP, yeast optimized in the Silver Lab BioFusion standard for fusion of protein domains.
Transcriptional terminator consisting of a 64 bp stem-loop.
Karl Holdar, Hampus Elofsson, Emil Marklund, Kristoffer Lundmark, Marcus Hong, Lovisa Pettersson, Ken Braech-Andersen, Theodor L??we
tyrosine ammonia-lyase (TAL) is an enzyme which catalyzes the formation of p-coumaric acid (aka p-hyroxycinnamic acid) from tyrosine. It belongs to the family of ammonia-lyases, enzymes that catalyze the deamination of amino acids [1]. P-coumaric acid is an important precursor in many metabolic pathways. TAL also has a secondary function as a phenylalanine ammonia-lyase (PAL) which catalyses the formation of cinnamic acid. The wild type RsTAL has a preference for tyrosine, however the mutations Met4 -> Ile, Ile325 -> Val and Val409 -> Met, can shift this preference to phenylalanine [2]. 1.Zhixiong Xue, Michael McCluskey, Keith Cantera, F. Sima Sariaslani, Lixuan Huang (2007) Identification, characterization and functional expression of a tyrosine ammonia-lyase and its mutants from the photosynthetic bacterium Rhodobacter sphaeroides. J Ind Microbiol Biotechnol 34:599-604 2. J.A. Kyndt, T.E. Meyer, M.A Cusanovich, J.J. Van Beeumen (2002) Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein. FEBS Letters 512 240-244
Earlier in iGEM eucaryot TAL has been used, however we used bacterial TAL for better compatibility with E.coli.
This part is made of constitutive promotor CMV and reporter Rluc with degradation tag PEST191.
This compositive part is cloned in vector BBa_J52017 (we have made it by ligating eucaryotic terminator in BioBrick vector pSB1AK3).
autoinducer inactivation enzyme aiiA (no LVA)
This is the standard GFP protein reporter (E0040) cloned with the prefix/suffix required for standard 25 assembly. It can easily be used to create fusion proteins through this method.
This part contains the AgeI and NgoMIV sites as part of it's prefix/suffix that allows it to be used in standard 25 assembly. It was synthesized through PCR oligo synthesis methods using the following primers: Forward (56.88 degrees) TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCCGTAAAGGAGAAGAACTTTTC Reverse (56.95 degrees) AATACTGCAGCGGCCGCTACTAGTATTAACCGGTCTTGTCGTCATCATCTTTATAAT
This part is similar to the part BBa_E0040 commonly used as a reporter.
Promoter (luxR & HSL regulated -- lux pL)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
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 consists of a TetR generator and a GFP with LVA tag under control of a TetR promoter. Normally the production of GFP-LVA is switched off. It can be switched on by the addition of tetracyclin.
This construct is used to screen the level of induction of the phiR73 delta activator on the PO promoter. Note that the I746352 does contain a RBS.
CinR (BBa_C0077) in complex with O3-C14:1-HSL (from CinI, BBa_C0076) binds to this promoter and activates transcription. This promoter contains the RBS: TGGAGG
The regulatory locus cinRI in Rhizobium leguminosarum conrols a network of quorum-sensing loci Lithgow, JK; Wilkinson, A; Hardman, A; Rodelas, B; Wisniewski-Dye, F; Williams, P; Downie, AJ MOL. MICROBIOLOGY 37(1): 81-97, 2000 Change log: the last 5 bases (gctaa) removed to allow for the correct spacing between the RBS (TGGAGG) and the start of the downstream gene after biobricks splicing.
unidentified/uncharacterized CinR dependent
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
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.
An RNA thermometer that theoretically switches on translation at 27 degrees Celcius and switched of translation below that temperature. Still to be tested.
Based on ROSE RNA thermometer sequences from Rfam database.
contains the J23060 loop on a tRNA. more on this later
Synthesizes 3OC6HSL, which binds to LuxR. 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 HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
An LVA tail (sequence: AANDENYALVA) was added to increase protein degradation. .
This part is designed to repress the expression of YFP via a cis-repressive element called cr5 which sequesters the ribosome binding site of YFP.
The cr element is a synthetic oligo which has been inserted into part I759009.
Protein generator converting TIPS to the protein cI from lambda. Used as the input section for Quad Part Inverters Q01150 and Q01151.
KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of yellow-orange light (540-585 nm). KillerRed is engineered from Aequorea victoria GFP (avGFP) to be phototoxic. It has been shown that KillerRed produces superoxide radical anions by reacting with water. Superoxide reacts with the chromophore of KillerRed, causing it to become dark, which ultimately gives rise to a bleaching effect. KillerRed is spectrally similar to mRFP1 with a similar brightness. KillerRed is oligomeric and may form large aggregates. This sequence is codon optimized for mammalian cells and has 14 rare proline codons for E. coli (CCC) and one rare arginine codon (AGA). Codon optimization should be taken into consideration if large amounts of the protein are required. Expression on a high-copy plasmid has produced detectable fluorescent signal, however.
This sequence is not codon optimized for E. coli.
Engineered from avGFP. Provided by the Bruchez lab at Carnegie Mellon
Jay Blumling, Debra Lin, Madeleine Sheldon-dante, Fred Tan, MIT SMUG
Coding sequence of GFP reporter protein under the control of CMV promoter.
RTX (repeats-in-toxin) is a polypeptide motif consisting of a repeating sequence of amino acids. It undergoes a conformation change in the presence of calcium which causes it to precipitate from solution. This is the shorter (8-mer) version of this part, and is in Freiburg format. This allows RTX to be fused to itself to create a longer RTX chain, or to other proteins which can then be precipitated from a solution. This is useful for the purpose of protein purification, or alternatively to remove an unwanted protein from a solution.
We ordered PCR primers to isolate RTX from its original construct. Due to the repetitive nature of this construct, we found that there were multiple binding sites for the forward primer which resulted in two products. This is the shorter PCR product, with only 8 repeats, whereas the original construct had 17.
medium RBS, YFP (-LVA), double terminator
beta-glucosidase gene bglX (chu_2268) from Cytophaga hutchinsonii
This is a beta-glucosidase gene (bglX, locus chu_2268) from the genome sequence of the cellulose-degrading Gram negative bacterium Cytophaga hutchinsonii.
Genome sequence of the cellulose-degrading Gram negative bacterium Cytophaga hutchinsonii.
Yellow Fluorescent Protein derived from A. victoria GFP. This fluorescent protein is cloned from the pZE12-YFP plasmid used by Elowitz (see reference). The original sequence was made by the The Yeast Resource Center (YRC) based at the University of Washington in Seattle, Washington.
This fluorescent protein was cloned from the pZE12-YFP plasmid used by Elowitz (see reference).
Elowitz, M. B., A. J. Levine, et al. (2002). "Stochastic gene expression in a single cell." Science 297(5584): 1183-6.
enhanced yellow fluorescent protein derived from A. victoria GFP
I746100 (AIP sensor infrastructure - agrC + agrA with RBSes) + B0015 (Terminator)
This part is simply the AIP sensor infrastructure ([http://partsregistry.org/wiki/index.php/Part:BBa_I746001 I746001]) from the agr system with a standard registry terminator ([http://partsregistry.org/wiki/index.php/Part:BBa_B0015 B0015]) downstream. The original part was made in order to transfer the S. aureus oligopeptide-based quorum sensing system into a BioBrick-compatible signalling mechanism. In the natural system, the signalling oligopeptide (termed AIP) is made from AgrD by the membrane-located enzyme AgrB. It is then detected by the membrane-located AgrC, which phosphorylates AgrA which then has DNA-binding activity and upregulates transcription of the promoters termed P2 and P3 in the agr locus. There are four known variants of AIP with different molecular structures and cross-inhibitory activity; this BioBrick generates group I AIP. This part contains the coding regions for group I agrB and agrD (in that order) from S. aureus strain NCTC8325, prefixed by the B0034 RBS and separated by a SaII site.
The original sequences for agrC and agrA were taken from the S. aureus Sequencing Project (results for strain NCTC8325) at Oklahoma University (http://www.genome.ou.edu/staph.html). The sequences were then codon-optimised for E. coli using GeneDesigner from DNA2.0 (http://www.dna20.com), and then synthesised by that company.
This part is an intermediate to the protein generator BBa_45200.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
Derived from luxICDABEG operon terminator of Vibrio fischeri AF170104 .
pbad promoter driving 6-his tagged mut3 GFP
This part is used for purification of mut3 GFP via its 6-his tag. It is driven by the arabinose - sensitive pbad promoter. Induction should be with 1mM arabinose for overnight growth of a 50ml (or more) culture for purification the next day.
The part was assembled from intermediates via standard assembly.
Overlap extension of synthetic oligonucleotides
CMV + Rluc is a composite part made of constitutive promotor CMV and Rluc, which acts as a reporter.
This protein domain can only be used within the TAL-effector-toolkit designed by the IGEM2012 Team Freiburg. The codon usage was modified to reduce homology in the sequence. A silent point mutation was inserted into the BsmBI restriction site in the chloramphenicol gene.
This Part was synthesized and flanked with BsmBI-restriction sites.
Lac operator with CFP reporter (without LVA tag) [R/Lc-]
Christopher Batten, Victoria Chou, Kenneth Nesmith
This part has been shown to repress the expression of YFP via a cis-repressive element called cr7 which sequesters the ribosome binding site of YFP.
This construct is used to screen the level of induction of the P2 ogr activator on the PLL promoter. Note that the I746350 does contain a RBS.
''dxs'', from ''Escherichia coli'' JM109, encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses the first step in the biosynthesis of terpenoids. Overexpression of ''dxs'' has been reported to increase yields of carotenoids and other terpenoids [add reference here]. ''lims1'', from ''Citrus limon'', encodes limonene synthase, which produces (+)-limonene from farnesyl diphosphate (an intermediate in the mevalonate pathway). (+)-Limonene is a component of lemon scent. ''appY'', from ''Escherichia coli'' JM109, encodes a transcriptional regulator related to anaerobic energy metabolism. Overexpression of the gene has been reported to result in enhanced lycopene production by ''E. coli'' (Kang ''et al.'', 2005). (All three genes are individually preceded by a ribosome binding site.)
The order of the genes in the operon corresponds to our perceived importance of each gene, bearing in mind that translation is more efficient of the genes early in the mRNA.
''dxs'' and ''appY'' are both from ''E. coli'' JM109 genomic DNA. ''lims1'' is from ''Citrus limon'' genomic DNA.
Signal sequence for myristoilation fused with proteolytic cleavage site for TEV protease and mCerulean, all under control of CMV promoter.
This part has been shown to repress the expression of YFP via a cis-repressive element called cr8 which sequesters the ribosome binding site of YFP.
The 434 cI repressor protein coding sequence is a 710 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the 434 regulatory sequence, BBa_R0052. The sequence contains a LVA tag for faster degredation and has no RBS.
References (unparsed) here: Nikolnikov,S., Posfai,G. and Sain,B. The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434. Gene 30 (1-3), 261-265 (1984) References (unparsed) here: Nikolnikov,S., Posfai,G. and Sain,B. The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434. Gene 30 (1-3), 261-265 (1984)
RBS (Elowitz 1999) -- defines RBS efficiency
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
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
Chloramphenicol resistance gene including its native promoter and ribosome binding site. Confers resistance to 25 ug/mL chloramphenicol. {pSB1A2-Bca1016}
PCR ca1016F/R on pKD3 (934 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Products are Bca1015 and Bca1016 ---- ca1016F Forward EcoRI to biobrick CAT of pKD3 gacttgaattcgcggccgcttctagaggaataggaacttcatttaaatg ca1016R Reverse SpeI to biobrick CAT of pKD3 ccgctactagtggcgcgcctacctgtgacgg
The p22 cII regulatory region sequence is a 97 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. p22 cII repressor protein, BBa_C0053, binds to it. This segment contains O-R1, O-R2, a fragment of O-R3, the -35 of P-RM, and P-R (-10 and -35 from Tom Knight)
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.
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor). dnMyD88 is linked to GFP (acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-GFP was made by using PCR overlap extension technique. This part is cloned in BioBrick vector pSB1AK3. Original sequence has PstI restriction site so we have to introduce a mutation without affect on amino acid sequence.
This is one of the constructs used to characterise superfolder GFP (see I746916 part description for source and other information about this GFP variant)
This part was created by standard assembly from construction intermediates: I746910 was put together with I746916:B0015
GFP reporter for RHS of library test constructs
This part will be suffixed to a promoter.RBS library. The purpose of the experiment is to make a first pass at a library-based construction step to identify efficiency issues and also to examine maxPOPS/RIPS level.
This part was built by Jen as an intermediate to their nuts and bolts parts.
A construct to make sure that luxR and effectively be coupled to the tetR inverter and shut down expression of GFP when AHL is present.
This part has been shown to repress the expression of YFP via a cis-repressive element called cr4 which sequesters the ribosome binding site of YFP.
Tet operator with CFP reporter (with LVA tag) [R/Tc+]
PenP operator from Bacillus Lichenformis. Two operator sites are negatively regulated by PenI (C0074).
AHL inducible production of hydrogen peroxide
This part produces pyruvate oxidase (K137069) in response to detecting acylhomoserine lactones.
The luxR reciever, RBS and double terminator are all from the registry. Pyruvate oxidase (spxB) is from a genomic PCR from Streptococcus pnumoniae R6.
engineered cyan fluorescent protein derived from A. victoria GFP
R6K origin of replication requires a pir+ or pir116 strain for replication in the absence of a second origin. In most E. coli strains, this is a silent feature. {pSB1A2-Bca1011}
PCR ca1011F/R on pG80ko (445 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Product is Bca1011 ---- ca1011F Forward Biobricking of R6K GGACTgaattcgcggccgcttctagagtgattcgcacgggcccatg ca1011R Reverse biobricking of R6K ccgctactagtaGCAGTTCAACCTGTTGATAG
An RNA thermometer that theoretically switches on translation at 37 degrees Celcius and switched of translation below that temperature. Still to be tested.
This chromoprotein, cjBlue, naturally exhibits strong green/blue color when expressed. Compared to amilCP(BBa_K592009) and amilGFP(BBa_K592010), the color development is slower. On agar plates and in liquid culture, the color is readily visible to naked eye after 24-48 hours of incubation. The DNA was codon-optimized for expression in E.coli and synthesized by the Korean company Bioneer Corporation.
Codon-optimization for expression in E.coli necessary.
The RBS (BBa_B0034) was combined with cinr (BBa_C0077), cinl (BBa_C0076) and D-term (BBa_B0014) to create a single transcription control device for BBa_R0078
Comparing to BBa_I761000, double terminator is added in the end of this construct
Zhipeng Sun, Hetmann Hsieh, Jeffrey Lau, David Ramos
Composite of J36831 and J36834. KaiA and KaiC protein expression in the ratio 1:2 by amount or ~1:4 by mass.
Formed by J36831 /SP ligated with J36834 /XP. see http://openwetware.org/wiki/IGEM:Harvard/2006/Cyanobacteria for more information; antiquated name is "J36022".
Jonas Demeulemeester and Benjamien Moeyaert
Fuse the LVA tag to the T7 coding region
This part is designed as a temperature inducible RBS. Only designed so far.
The secondary structure is important to the function of these regions, but part of the wt secondary structure is destroyed by the scar. We've tried to alter the sequence so the predicted structure (through mfold and those kind of servers) is sort of conserved, but temperature sensitivity still has to be tested. If it doesn't work, possible solution might be the addition of a larger conserved part of the wt, which implies a small part of wt protein sequence as well.
This sequence is taken from the Bradirhizobium Japonicum (BA000040.) as the 5'UTR (ROSE) of a heat shock protein (prfA).
Predicted Stem Loop extending to startcodon
Promoter (lambda cI and luxR regulated -- hybrid)
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 .
dxs coding sequence encoding 1-deoxyxylulose-5-phosphate synthase
This is the coding sequence of dxs from Escherichia coli JM109. It encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses teh first step in the biosynthesis of terpenoids. Overexpression of dxs has been reported to increase yields of carotenoids and other terpenoids [add reference here].
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Fusion Protein of S-Layer SbpA and mCherry RFP
Fusion Protein of BBa_K525403 and BBa_J18932
This chromoprotein from the coral Anemonia majano, amajLime, naturally exhibits strong color when expressed.
Synthesized and codon optimized for E coli by GenScript.
enhanced cyan fluorescent protein derived from A. victoria GFP
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Cyan fluorescent protein (ECFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission.
BBa_E0022 cyan fluorescent protein is based on BioBrick part BBa_E0021. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0021</bb_part>.
Fusion Protein of S-Layer SgsE and mCitrine
Fusion Protein of BBa_K525303 and BBa_J18931
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.
Promoter (lacI regulated, lambda pL hybrid)
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
represillator of Elowitz and Leibler (2000)
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.
RBS.4 (weaker) -- derivative of BBa_0030
Weaker RBS based on Ron Weiss thesis. Strengths relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-3" in figure 4-14 of thesis). 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
This parts produces GFP in response to detecting an acylhomoserinelactone (AHL). It was made to check that the RBS behind the luxR reciever was functional.
double terminator (B0012-B0011), reversed
Part is composed of NfΚB promoter (recognation of this promoter for initiation of transcription depends on the presence of nuclear factor kappa B), TIR domain of Traf6 which is linked with 6 amino acid long linker to GFP.
The second portion of the RFP gene, after the HixC insertion point.
uniCAS RNAimer multiple targets to VEGF 2 and VEGF 3
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">
RhlR can sense N-butyryl-HSL in the media and can activate transcription from R0071. It can be used as part of a receiver device.
CRISPR-Cas is a bacterial immune system that remembers and targets foreign viral DNA by storing DNA sequences, or spacers, between clustered regularly interspaced short palindromic repeats (CRISPRs). RNA transcripts of the spacers are then used to sense homologous DNA, which is cleaved by CRISPR-associated (Cas) proteins. This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites, which allow for the easy cloning of new spacers.
Mutagenesis to remove internal EcoRI site
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087) preceded by an E. coli synthetic ribosome binding site (rbs). 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.
rbs: Escherichia coli JM109 genomic DNA. crtI: Pantoea ananatis genomic DNA
Removed LVA tag from BBa_C0052 via PCR. In pSB1AK3 currently.
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
Limonene synthase gene with ribosome binding site
Ribosome binding site attached to Limonene synthase gene. Limonene synthase enzyme produces (+)-limonene from farnesyl diphosphate (intermediate in mevlonate pathway). (+) Limonene is a component of lemon scent.
several codons were altered silently to remove several forbidden restriction sites. The N-terminal region (plastid targeting sequence) was removed, removing the first 51 amino acids and replacing codon 52 with the start codon ATG. LIMS cDNA was used as genomic DNA contains introns.
Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases, J Lucker, et al, Eur. J. Biochem. 269: 3160???3171 (2002) Analysis of Lemon and Bergamot Essential Oils by HPLC with Microbore Columns, M Benincasa, et al, Chromatographia 30(5/6):271 (1990) Citrus fruit flavour and aroma biosynthesis, L Sharon-Asa, The Plant Journal, 36:664 (2003)
We improve the function of a BioBrick Part: AhpC promoter([http://parts.igem.org/Part:BBa_K362001 K362001])designed by [http://2010.igem.org/Team:KIT-Kyoto/Parts 2010 KIT-Tokyo team] into four versions.On PartRegistry, the complex part(according to [http://ecocyc.org/ECOLI/new-image?object=EG11384 Ecocyc]) composition contains hybrid promoters, shared TFBS (Transcription Factor Binding Site), and reverse promoter DsbG. This part contains 3 promoters: AhpC1, DsbG, AhpC2, 2 TFBSs for OxyR, and coding sequence in the initiate part([http://parts.igem.org/Part:BBa_K362001 K362001])was deleted.
We delete the dsbG coding and following sequence next to dsbG reverse primer, making the functional sequence shorter, so that PCR can be more easy and efficient.
Protein generator converting TIPS to the protein cI from 434. Used as the input section for Quad Part Inverter Q01520.
Reporter GFP-linker-PEST191 under NF-kappaB inducible promoter. Used for detection of promoter activity and degradation of GFP.
Promoter has a NotI restriction site. We did not alter DNA sequence, because this site did not affect our part construction and function of promoter. This part is cloned in plasmid with terminator BBa_J52017.
NF-kappaB - GFP-PEST191 - already described (see BBa_J52028)
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Yellow fluorescent protein (EYFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. Annotated mutation cause a Q81L mutation that appears to not be near the active site.
BBa_E0032 yellow fluorescent protein is based on BioBrick part BBa_E0031. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0031</bb_part>
tet-regulated cro (Strong tetR promoter)
cI repressor from E. coli phage lambda (+LVA)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator (BBa_R0051).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 BBa_C0051 cI repressor is based on the cI repressor from the Elowitz's repressilator. It has been modified to include a rapid degradation LAA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
Medium RBS based on Ron Weiss thesis. Strength considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-1" in figure 4-14 of thesis). 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 Cho
c2 repressor from Salmonella phage P22 (+LVA)
The P22 c2 repressor protein coding sequence is a 720 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the P22 c2 regulatory sequence, BBa_R0053. The sequence contains a LVA tag for faster degredation.
References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000) References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000)
Vinay S. Mahajan, Brian Chow, Peter Carr
Generic spacer for ensuring a 70 bp distance between the end of the suffix of the BioBrick part containing the double terminator and the prefix of the BioBrick part containing the promoter of the new gene. Please, use the AlignX function of Vector NT to check for homology with the components in your plasmid before using this spacer.
The size of the spacer was choosed to meet the minimum length of a sequence that can be queried using the BLAST search engine. However, subsequences of it can be used to design shorter spacers. The sequence was selected from many more sequences randomly generated using the Random DNA Generator engine; the GC% parameter used as input was 50%. The sequences were selected based on the following constraints listed in their order of importance: the absence of any putative promoter regions, a low degree of homology with the Elowitz plasmid (whose components are widely used in our designs), no homology with other E.coli sequences as shown by BLASTN search results and the presence of a number of TAA stop codons. The second constraint was the most stringent leading to the elimination of most sequences. DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA. Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur): T->A @ 85 T->A @ 42 C->T @ 79 A->T @ 64 A->T @ 31 T->A @ 34 C->A @ 37 Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences. In the process of selecting the best sequence it appeared that a good alternative sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA. The sequence contains no putative promoters and shows moderate homology with the 5' end of the Ampicillin resistance gene. However a strong promoter sequence starts 12 bp downstream of this sequence, and therefore the sequence presented above was preferred. The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.
Randomly generated and optimized for several parameters (see Design notes).
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
this device consists if a TetR generator and a GFP under the control of a TetR regulated promoter. Normally the production of GFP is switched off. It can be switched on by the addition of tetracyclin (or anhydrotetracyclin).
autoinducer inactivation enzyme from Bacillus; hydrolyzes acetyl homoserine lactone
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita D. Marinescu and Alexander D. Wissner-Gross
Coding region for the autoinducer inactivation enzyme A (aiiA) LVA tagged. The gene was originally isolated from Bacillus sp. 240B1 and it encodes an enzyme that catalyzes the degradation of N-acyl homoserine lactones (AHLs)--quorum sensing autoinducers.
References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 BBa_C0060 insert contains open reading frame (nucleotides 49-801) of the GeneBank sequence AF196486 followed by the LVA tag and two double stop codons inserted in the BioBrick prefix and suffix flanking regions. The original stop codon was TAG and in the present sequence it was substituted by TAATAA.
AF196486 from Bacillus sp. 240B1 putative metallohydrolase (aiiA) gene. Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000).
Apoptin is a protein that is originally isolated from the chicken anemia virus(CAV). It has been found that expressing this gene can induce apoptosis in a broad spectrum of cancer cells but not in normal cells. Hence it is a very powerful tool in treating cancer. Fusion the TAT protein with apoptin can even achieve by-stander effect, and we try to use it in the most simple device of our project. We have successfully expressed it in Hep G2 cell lines and observe a brilliant apoptosis effect.
Although it has been try in a broad spectrum of cells and prove to be safe to the normal cells, it still has not been tried in ALL normal cells. So before using it to treat certain kinds of cancer, every team need to ensure that it want hurt the specify normal cells involve in the project. And it is a protein contain 121 amino acid that may big enough to induce immune reaction(although in the experiment of mouse it has not been reported.)
Promoter, Standard (lambda cI regulated)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross Drew Endy
[Note: This is the same part as R0051 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Modified to comply with BBa_S0001: TTGACA-17N-GATACT Incompatible with host expressing cI repressor.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
An RNA thermometer that theoretically switches on translation at 32 degrees Celcius and switched of translation below that temperature. Still to be tested.
FepA L8T Mutant - Large Diffusion pore for E. coli outer membrane.
FepA is an outer membrane receptor protein for Escherichia coli involved in the uptake of the ferric enterobactin siderophore. This part is a deletion mutant of the wild type FepA, which has been shown to convert the protein into an open diffusion channel, similar to a porin. It allows larger molecules through than normally would be permitted.
The original mutants had an EcoR1 restriction site within their coding sequence. This had to be removed via site directed mutagenesis, using a megaprimer based method.
The mutants were developed by Dr Salete Newton working in Professor P. Klebba's lab at the university of Oklahoma. They were kindly sent to us by Professor Klebba.
This chromoprotein naturally exhibits strong color when expressed.
This construct is synthetic. It was synthesized from a template where the consensus sequence for the "color dependancy" in order to obtain a chromoprotein with a unique yellow color.
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.
Protein generator converting TIPS to the protein cII from p22. Used as the input section for Quad Part Inverters Q01530.
T7 RNA Polymerase with N-terminal UmuD degradation tag.
This part is different from the simple composition of the tag and the polymerase, as there cannot be a stop codon in the scar.
Part composed of the T7 RNA Polymerase and the N-terminal degradation tag, but with no scar (no stop codon)
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor), which was our main target for modifying the signaling path. dnMyD88 is linked to Rluc (which acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-Rluc was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
This part is an intermediate to the protein generator BBa_45300.
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Incompatible with host expressing cI repressor.
rbs+dxs (1-deoxyxylulose-5-phosphate synthase)
This is the coding sequence of dxs from Escherichia coli JM109 with added ribosome binding site (rbs). It encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses the first step in the biosynthesis of terpenoids. Overexpression of dxs has been reported to increase yields of carotenoids and other terpenoids [add reference here].
This part accepts POPS to produce ECFP under the medium RBS B0032
Fusion Protein of S-Layer SgsE and mCerulean
Fusion Protein of BBa_K525303 and BBa_J18930
This part has been shown to repress the expression of YFP via a cis-repressive element called cr2 which sequesters the ribosome binding site of YFP.
Binding region for LasR protein (positive regulation)
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
Contains both a BbsI and a BstXI restriction site. When cut with both enzymes, it leaves an EcoRI and a PstI site.
70 bp from Eco to Spe or Xba to Pst, to allow for gel extraction. The middle portion is largely noise, intended as filler to raise the size of the part.
This construct is used to screen the level of induction of the PSP3 pag activator on the PO promoter. Note that the I746351 does contain a RBS.
Coding region for the HK022 bacteriophage cI protein. cI binds to the HK022 pR regulator (BBa_R0050). It represses transcription of the protein encoded by the sequence 3' to the pR region. This coding sequence does not contain a RBS.
References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. Derived from STHK022N . Response from John Little (Arizona) regarding the start of HK022 cI From: <jlittle@email.arizona.edu> Date: Tue Jan 21, 2003 4:39:21 PM US/Eastern To: "Drew Endy" <endy@MIT.EDU> Subject: RE: hk022 cI start (naive question)? Hello Drew and Michael, I seriously doubt that the extra 27 aa are part of CI. It doesn't make sense in terms of the biology, for sure. In any case, the protein we characterized starts where Carlson and Little stated; as I recall, oR3 partially overlaps the start of cI. I have a vague memory of some possibly interesting biology, having to do with multicopy plasmids. I don't recall the findings themselves. Anyway, one possible explanation was that this extra N-terminal addition was made (e.g. from the pRE promoter, or from a message that arose from transcription around the entire plasmid), making a protein with altered functions. We never followed it up. Good luck John -- Original Message -- Date: Sun, 19 Jan 2003 15:26:26 -0500 Subject: hk022 cI start (naive question)? Cc: elowitm@rockefeller.edu To: jlittle@u.arizona.edu From: Drew Endy <endy@MIT.EDU> Hi John, I'm sitting here with Michael Elowitz and we're working through the sequence for HK022 cI. We noticed that the annotation from NC_002166 includes an "extra" 81 base pairs upstream of what we thought of as the actual start. It looks like this extra DNA extends into the PR regulatory region. We're wondering what's going on here? I'm guessing this is well documented somewhere. Thanks for any pointers/info! Drew It is unknown whether there is cross-talk between this repressor and Lambda cI regulatory region, 434 cI regulatory region and P22 regulatory region.
Receptor interacting protein 3, also a serine/threonine kinase that play an important role in cell apoptosis and necrosis. It can induce necrosis via the interaction with RIP 1.
Although RIP 3 can also induce apoptosis, it mainly induces necrosis via interaction with RIP 1. So the mammalian circuit that aid to use in clinical should use tightly control expression device.
Plac+rbs+glgC (ADP-glucose pyrophosphorylase)
This is the coding sequence of glgC from ''Escherichia coli'' JM109 (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K118015 BBa_K118015]) with a ribosome binding site coupled to an upstream Lac promoter and ''lacZ''' as a method for testing expression.
This sequence is taken from the Listeria Monocytogenes (EU372032.). It's the 5'UTR of a TF induced at 37 degrees.
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the lac repressible R0011.
CD4 - Cub fused with GFP signal under cmv promoter.
Ligation of two biobricks(BBa_I712085, BBa_J52642).
This part is an intermediate to the protein generator BBa_45220. This part is almost identical to BBa_J45199 except that it uses B0032 as its RBS (instead of B0030).
I746101 (AIP sensor infrastructure) + I746105 (Inducible P2 + GFP reporter)
This part is the AIP sensor infrastructure ([http://partsregistry.org/wiki/index.php/Part:BBa_I746101 I746101]) from the agr system with an AIP-inducible promoter P2 and GFP reporter ([http://partsregistry.org/wiki/index.php/Part:BBa_I746105 I746105]) downstream. A constitutive/inducible promoter may be added upstream of this part, which will cause production of the components of the signal transduction pathway (AgrC and AgrA); when phosphorylated AgrA is present, the AIP-sensitive promoter (P2) is activated and GFP ([http://partsregistry.org/wiki/index.php/Part:BBa_E0840 E0840]) is output. The original part was made in order to transfer the S. aureus oligopeptide-based quorum sensing system into a BioBrick-compatible signalling mechanism. In the natural system, the signalling oligopeptide (termed AIP) is made from AgrD by the membrane-located enzyme AgrB. It is then detected by the membrane-located AgrC, which phosphorylates AgrA which then has DNA-binding activity and upregulates transcription of the promoters termed P2 and P3 in the agr locus. There are four known variants of AIP with different molecular structures and cross-inhibitory activity; this BioBrick generates group I AIP. 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 activity of agr P2 promoter about 50-fold.
It is a composite part, coposed of NF-kB dependent promotor and dominant negative human MyD88
added different promoter RBS to increase expression
Trevor Kalkus, Gordon Wade, Alissa Greenberg
The E. coli CRISPR system uses the CASCADE complex to target invasive DNA. CasA is the first subunit of the CASCADE complex and is responsible for the initial identification of invasive DNA. This part can be used with casBCDE to create the full CASCADE protein complex.
We had to perform mutagenesis twice to remove PstI cut sites.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator <bb_part>BBa_R0010</bb_part> and PLlac01 hybrid regulator <bb_part>BBa_R0011</bb_part> and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degredation tail (LVA) has been added to improve the High to Low performance of this part.
References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] Sequence taken from the repressilator of Elowitz and Leibler (2000). The obtained sequence was compared to the wild-type sequence for LacI obtained through a database search. The sequence had been modified from the wild-type in that wild-type GTG start was changed to an ATG start (note, actual ORF in E.coli has several GTG starts it would seem). The LVA tag has been added for quicker degradation. Incompatible with systems containing LacI, lactose, or IPTG.
tetracycline repressor from transposon Tn10 (+LVA)
Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). 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 Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). 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 Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 BBa_C0040 TetR Protein is based on the TetR sequence from Elowitz's repressilator. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212)
GFP-AAV [Pst1 site in earlier version knocked out]
This part has been shown to repress the expression of YFP via a cis-repressive element called cr6 which sequesters the ribosome binding site of YFP
Fusion Protein of S-Layer SbpA and mCerulean
Fusion Protein of BBa_K525403 and BBa_J18930
This device can generate luxI. luxI is an enzym that can catalyse the production of HSL. When no key is present, there will be no lacI and therefore luxI will be produced. However, when key is present, lacI will be produced and this wil repress the production of luxI.
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
Firefly luciferase - luciferase from Photinus pyralis
Firefly luciferase is luciferase from Photinus pyralis. By oxidation of substrate luciferin light is produced. Luciferase thus acts as a reporter protein when connected to other proteins or promoters.
Part is cloned into pSBAK3 vector with terminator.
This firefly luciferase comes from Promega PGL3 vector.
The first two nucleotides of the GFP start codon were removed to create this fusion brick.
The GFP fusion brick was derived from the GFP BioBrick, [http://partsregistry.org/Part:BBa_E0040 BBa_E0040].
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). 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 Brian Chow
Luis Mario Leal Garza & Luis Fernando Camarillo Guerrero
This secretion device includes all the necessary elements (linker-HlyA secretion signal peptide and the expression cassette for HlyB and HlyD) for the secretion of expression cassettes cloned in the 5??? terminal of the device. This secretion device is usable by Silver standard fusion in its 5??? end for proteins with no stop codon. Codes for both the proteins HlyB and HlyD (native from uropathogenic strains of E.coli), which, along with the constitutively expressed TolC protein, form a secretion channel. This secretion system recognizes proteins tagged with the C-terminal secretion signal peptide HlyA, and secretes them to the exterior of the bacteria.
HlyA C terminal part has a 5' section compatible with Silver Fusion standard. Downstream, HlyB and HlyD are both under the AraC/pBAD promoter to be induced by arabinose.
The HlyB and HlyD sequences come from the parts registry, while the HlyA from the registry had mayor problems, so we took it from the genomic sequence.
This part is made of constitutive promotor CMV and GFP-linker-PEST191 which acts as a reporter linked to degradation tag (PEST191).