Encodes a stable non-specific ribonuclease in ''Bacillus Subtilis''. It is used in conjungtion with ''mazE'' in ''Bacillus Subtilis'' to provide a toxin-antitoxin in various stressful conditions. When transcribtion of both genes is turned off (as both are under contol of same promoter) ''mazE'' will be degraded faster than ''mazF''. There is then no inhibiton of ''mazF'', killing the cell.
Sequence isolated based upon the work of Pellegrini, O., Mathy, N., Gogos, A., Shapiro, L., and Condon, C. (2005) The ''Bacillus subtilis'' ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor. ''Mol Microbiol'' 56: 1139???1148.
This biosafety device for plants consists on two submodules: a male sterility submodule and an identity preservation submodule. The male sterility submodule consist on a barnase specifically expressed in anthers under the regulation of the TA29 tapetum-specific promoter. As result, pollen from these plants is not fertile. The identity preservation submodule expresses the blue chromoprotein AmilCP, so that plants can be visually differentiated from non-transgenic plants.
The assembly was performed by GoldenBraid 2.0 assembly. The barnase was amplified by PCR from [http://parts.igem.org/Part:BBa_J72173 Part:BBa_J72173] and a mutation in T105P was introduced.
TA29 promoter [http://parts.igem.org/Part:BBa_K1554000 Part:BBa_K1554000] Barnase [http://parts.igem.org/Part:BBa_J72173 Part:BBa_J72173] AmilGFP [Waiting for Norwich team part name in registry] P35s [http://parts.igem.org/Part:BBa_K1537015 Part:BBa_K1537015]
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=66503&to=66997&view=gbwithparts
The lysozyme from enterobacteria phage T4 degrades peptidoglycan layer.
High expression of lysozyme only is toxic to bacteria.
A SV40 nuclear localization signal was added to the phototoxic protein KillerRed.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
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.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
This part is made from the 3 Biobricks previously describe : R0011 - K228001 - B1006
This part is composed of a tRNA amber suppressor, supD (K228001), which is under the control of pLac (R0011). A terminator is added (B1006, but the sequence is not the exact one, it's not a problem here). This part is used to allow the expression of gene that have one or many amber codon instead of a Serine. Adding IPTG could be usefull when there are to many amber codon in the cell.
The promoter is the last part added, meaning that from the previous intermediate (soon available), we will be able to change the promoter easily.
V. fischeri <genbank>AF170104</genbank>
In complex with HSL, LuxR binds to the Lux promoter, activating transcription from Pr <bb_part>BBa_R0062</bb_part>, and repressing transcription from Pl <bb_part>BBa_R0063</bb_part>. The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux activator, LuxR complexed to HSL. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
2 silent point mutants were introduced in the coding sequence to remove internal XbaI and PstI sites. Mutation sites were chosen to replace codons commonly used in E. coli with codons used at a similar frequency.
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
CcdB is a toxin that poisons and kills E. coli by inhibiting the DNA gyrase and inducing double strand breaks. CcdB activity can be inhibited by the CcdA antitoxin. This gene is under the control of a galactose inducible promoter
CcdB is toxic so it has to be expressed in conjunction with CcdA or not on a galactose-containing medium.
>gnl|ECOLI|EG10525 lacI "PD00763" (complement(366734..365652)) Escherichia coli K-12 substr. MG1655 http://ecocyc.org/ECOLI/sequence-rc?type=GENE&object=EG10525
Coding region for the LacI protein. LacI binds to the lac promoter BBa_R0010 and inhibits transcription. IPTG binds LacI and inhibits its function, and thus promotes transcription from the lac promoter.
This colicinE1 cassette is derived from the naturally existing colicinE1 plasmid out of the E. coli strain JC411.
This colicinE1 operon contains all three important genes for colicinE1 expression and release. The colE1 gene codes for the lethal colicinE1 protein. The gene for the colE1 immunity protein located on the reverse strand is responsible for the immunity of the producing host bacteria to the colicinE1 and is therefore controlled by a constitutive promoter. Expression of the kil protein leads finally to the lysis of the host bacteria and thereby to the release of the colicinE1.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
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.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
Lysis gene (promotes lysis in colicin-producing bacteria strain)
This lysis gene encodes for the lysis protein in colicin-producing strains of bacteria. Once activated, it causes the host cell to lyze. It also removes the immunity protein out of colicin, and hence, activates the endonuclease activity of the colicin.
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=160221&to=160877&view=gbwithparts
Holins from T4 bacteriophage assemble together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer.
Making composite part of rbs.holin is problematic unless antiholin is expressed in bacteria together. Antiholin binds holin and inhibits the formation of holin.
Biofilm formation device. yddV gene with the IPTG inducible promoter.
Gene yddV is under the regulation of Ptrc promoter (IPTG-inducible). yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bis-(3???-5???)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentrations of c-di-GMP promote bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
cI repressor from E. coli phage lambda (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
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.
Colicin E2 Immunity protein under pLac promoter (BBa_R0011) with repressilator RBS. This part can be used to confer immunity against the colicin E2 activity protein by induction with IPTG.
We selected the repressilator RBS and pLac to drive production of Colicin E2 Immunity protein
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">
A Kill Switch with "memory" time repressed by IPTG
Name Number Description CI coding part BBa-C0051 Coding cI Promoter 1 BBa-J23116 Weak constitutive promoter Promoter 2 BBa-R0010 LacI repressible promoter Promoter 3 BBa-R0051 CI repressible promoter Table 1 Biobricks used in the design
We plan to establish a new E.Coli strain without lacI and MazEF system gene to avoid the inference of cell itself. We will test cI background expression level to see if we need to knock out cI. As is shown in Fig.1, Fig.2, Fig.3 and Fig.4, in the ???Stable State???, before poured into the soil, the E. Coli fertilizer is cultured in the medium with IPTG. Promoter 1 is a weak constitutive promoter, so lacI is transcribed and translated at a considerate low level. Therefore, high concentration of IPTG, which binds LacI and changes LacI???s conformation, is able to inactive LacI and open the promoter 2. Then, CI can be highly expressed to repress promoter3. Finally, mazF is inhibited. After fertilizing, no more IPTG exists in the soil to bind LacI. As a result, LacI with a tag which stabilize the protein takes time to accumulate to a certain high concentration, which is the repression threshold of promoter 2, aiming to represses promoter 2 as well as the expression of cI. The time needed to finish this progress is the designed ???memory time???. During this process, E. coli takes its own responsibility to deliver Mo. And after that, it can be killed to reduce the pollution to environment. As is shown in table 1, all the biobricks in the design are from the top 10 most used parts of iGEM to guarantee the feasibility. Name Number Description CI coding part BBa-C0051 Coding cI Promoter 1 BBa-J23116 Weak constitutive promoter Promoter 2 BBa-R0010 LacI repressible promoter Promoter 3 BBa-R0051 CI repressible promoter Table 1 Biobricks used in the design In the future, we plan to model the ???memory time??? and do more wet-experiments to confirm its feasibility. As for the modeling, we will test the expression level of promoter 1, the minimal stand of LacI to repress promoter 2 and the degradation speed of LacI. On the other hand, we will test the background expression of CI after removing IPTG to check if the CI concentration is low enough to open the promoter 3. Then we will strive to make this system more effective.
This part is just for upload test. It is meaningless
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
This part was cloned from a plasmid containing the operon which was received from UC Berkeley.
This part contains the ColE2 operon in its entirety.
J23103 promoer - RBS B0034-RFP E1010 - Holin K112805 - endolysin K112806
A composite part comprising of a J23100 promotor and PA2652 translational unit encoding extracellular chemoreceptor
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 (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Part 1 of the Biosystem TetOR alive (pRha TetR alr)
Part 1 of the Biosystem M (pRha TetR alr)
kanamicin ressistance gene (promoter+CDS)
selection for cassette integration into E.coli BL21 genome
ColicinE1 Producer Controlled by 3OC6HSL Receiver Device
The constitutive expressed LuxR protein is able to build a complex with the AHL molecule. This complex function as transcription factor for the lux pR promoter and activates by this the colicinE1 production. This activation of the lux pR promoter can additionally lead to expression of the kil protein by read through of the terminator t1 and therefore to lysis of the cell and colcin release. The colicinE1 immunity protein (imm) lying on the reverse strand is expressend under a constitutive promotor.
Pascal Kraemer, Marika Ziesack, Kathrin Nussbaum and Andreas Kuehne
pCons-TetR-pTet-TevProtease Suicide Switch
This is a kill switch part of our engineering E. Coli. pTet is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog, aTc. In the presence of aTc TetR repression inhibited and Tev Protease produce by Ptet promoter. Tev Protease release Pexiganan from DAMP and E. Coli kill switch works succesfully.
This gene is mutated by the PCR quick change mutagenesis.
pSB1A2 have a double mutation in the 80th serine (TCC->TAG) of the APr gene. This plasmid need a tRNA amber suppressor to express this gene. So in case of horizontal gene transfer this gene won't be expressed and then the plasmid will be lost more easily.
2 oligo mutated on 2 positions were used for this purpose.
This is the intermediate of the K914000 part, so you can change the promoter easily.
The terminator is mutated, but it does not impact the function.
codes for protegrin-1 (anti-microbial peptide)
This sequence was derived from protegrin-1 found in porcine leukocytes. The website where we found the sequence is:
This is the coding region for protegrin-1.
This is a part used for coding soluble human guanylate cyclase subunit:Alpha 3.Alpha 3 unit interacts with a beta subunit to form the guanylate cyclase enzyme. Soluble guanylate cyclases(sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP and pyrophosphate. It is important for smooth muscle relaxation in the cardiovascular system. sGC contains an HNOX domain, which serves as a receptor for ligands such as nitric oxide, oxygen and nitrovasodilator drugs.There are several different isoforms of sGC subunit ,but the cardiovascular system abundant with alpha 3 unit and beta 3 unit. This part with a GFP reporter was then transfected into HEK293 cells by lentiviral Vector and in vivo green fluorescence signal was observed under fluorescence microscope, it meant that this part was transfected into HEK293 cells successfully.Then we used qRT-PCR to observe transcriptional level of alpha 3 unit.Finally we detected the cGMP concentration with Elisa Kit. And the results are as follow:
This part do not have promoter because the promoter we used in our project is EF1A which consists the restriction sites.So before using this part please add a promoter.
We find the information of Human guanylate cyclase??1,soluble, alpha 3 unit here：http://www.ncbi.nlm.nih.gov/gene/2982
Endolysin is a lysozyme which cleaves the glycosidic bond between the C-1 of N-acetylmuramic acid (NAM) and the C-4 of N-acetylglucosamine (NAG) in the bacterial peptidoglycan (PG). This leads to the complete lysis of the cell. Holin is a protein that assembles together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer. It is sourced from T4 bacteriophage. This composite part can be used to induce self-destruction with a regulatory device in the upstream region.
There is no RBS in the upstream region, so during the assemble procedure, this must be taken care of.
Gene sequences of endolysin and holin are gathered from the Registry.
P1003** Kan resistant gene with 2 Amber Codon
This part is made from the BBa_K914009 part from which I add a second mutation with the QuickChange Site-directed mutagenesis kit, Agilent.
The amber mutations avoid the expression of the P1003 gene even at usual concentration that prevent the expression of kanamycin resistance, although one mutation (BBa_K914009) permits some leakage in translation for this gene. This mutation is rescued with the tRNA amber suppressor supD (BBa_K914000). The idea here is to prevent the kanamycin gene resistance to be expressed in case horizontal gene transfer.
I have need to design the primers that will introduce a mutation from a serine that was closer to the TAG codon (either 1, 2 or 3 mutations, here it was 1 and 2 mutations for both mutations), and it is necessary to have the amber codon in the middle of the coding sequence, not in the beginning, to be not interpreted as a alternative start codon for instance.
alanine racemase (''alr'') under the control of the P''tac'' promoter
Alanin_Racemase (alr) under the control of the ptac-Promoter
Part 1 of the Biosafety-System Lac of Growth (pRha lacI alr)
Part 1 of the Biosafety-System L (pRha lacI alr)
long description long long long just for test
It is a repeat-spacer-repeat region designed according to E.coli's CRISPR/Cas system. It also includes a T7 promoter and T7 terminator. THe spacer is designed to target on a segment of non-coding-sequence in E.coli's genome. This biobrick can be transformed into E.coli cells to hopefully construct a suicide system with E.coli's CRISPR/Cas system.
The repeat-spacer-repeat region design should be consistent with the operating principles of E.coli's CRISPR/cas system.
The 'repeat' sequence in CRISPR/cas system of E.coli strain K12.
DNA linker region between lux pR promoter BBa_R0062 and ribosome binding site pTrc99a (BBa_) for the ColicinE1 Producer Controlled by 3OC6HSL Receiver Device (BBa_).
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
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 this regulator. This part is incompatible with environments containing lactose or lactose analogs.
Alanine-racemase extracted from genomic DNA from E. coli KRX. Used for our biosafety project.
Ligation via Gibson-assembly, so the primer had to have certain extensions
Tev Protease, single S219V mutation in the internal cleavage site
TEV protease (also called Tobacco Etch Virus nuclear inclusion a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV).The consensus for these native cut sites is ENLYFQ\S where ???\??? denotes the cleaved peptide bond. One of the main uses of this protein is for removing affinity tags from purified proteins. The reason for the use of TEV protease as a biochemical tool is its high sequence specificity. This specificity allows for the controlled cleavage of proteins when the preference sequence is inserted into flexible loops. It also makes it relatively non-toxic in vivo as the recognized sequence scarcely occurs in proteins.[1]
This part contains an additional BamHI restriction site at beginning of Tev Protease sequence and a XhoI restriction site at the end.
The single mutation avoid the expression of the P1003 gene, that prevent the expression of a kanamycin resistance. This mutation is rescue with the tRNA amber suppressor supD (BBa_K914000), or any genotype relative to sup44 . The idea here is to prevent the kanamycin gene resistance in case horizontal gene transfer. Actually it would be better with another mutation.
I made the single point mutation with a quick change directed mutagenesis kit.
Different strength of green fluorescence will be generated determined by the concentration of IPTG.
Double terminator consisting of BBa_B0010 and BBa_B0012
CRISPR-Cas9 contaiment device repressed by arabinose and tryptophan
This is a prototype containment device utilizing CRISPR-Cas9 system which aims to control accidental leakage of GMM once it escape from medium containing arabinose and tryptophan. The device consists of two modules, firstly a arabinose repressible cas9 expression module and a tryptophan repressible gRNA expression module. Under the presence of sufficient amount of arabinose and tryptophan the device remain silent, however when bacteria escape to environment without arabinose and tryptophan, Cas9 and a gRNA targeting DNA polymerase III alpha subunit will be expressed. Subsequent cleavage of the targeted sequence may hinder DNA replication and
Rnase Ba (Barnase) from Bacillus amyloliquefaciens
Rnase Ba (Barnase) from Bacillus Amyloliquefaciens
The device consist of temperature promotor and hokD coding gene.
This device is a combination of two parts submitted by previous igem teams that one （BBa_K608351） is a temperature-regulated promoter followed by the other （BBa_K1497008 ）that a gene coding for a protein hokD which induces cell lysis. The promoter we chose won???t start translation until the temperature is raised to 37℃. And at 42℃ can we get the peck efficiency of expression. The promoter is in fact a composite sequence that preceding the regulated promoter is the control sequence which can synthesis λ cI repressor. λ cI repressor binds with the promoter sequence when environment temperature is 28~30℃ while at 42℃, the protein denatures and falls off by which initiate the transcription. We use the temperature-regulated promoter to express hokD gene. The hokD gene of??E. coli??transcribes for a small polypeptide that causes cell death by elimination of vital cell wall functions. In short , our device can induce cell lysis when temperature is raised to 42℃. To deliver the antimicrobial peptides in the right place and at the right time, we designed and constructed this temperature-regulated device. When put under the sole of feet, our peptide-expressing E.coli cells in the insole would be incubated at a warmer environment due to body temperature. We use this promoter to control the synthesis of hokD for the raised temperature will promote the translation of hokD which following the promoter.
We use pSB1C3 vector to express this device for its more avaliable.
we acquired that two parts BBa_K608351 and BBa_K1497008 we based on on igem distribution plates and constructed the composite device through molecular biology technique.
Receiver cell, T4 lysis device is under pLas.
We got BBa_K772004 from 2012 Fatih Medical team.
LasR Device:K772004 Enterobacteria phage T4 Lysis Device - no promoter: K112808
Part 2 of the Biosafety-System L with GFP (dplac GFP)
tetracycline repressor from transposon Tn10 (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
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.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
A DNA cassette that undergo homologous recombination into E.coli genome that contains homologous regions at each end, Kanamicin resistance gene (KanR) and death operon containing Lambda holin and lysozime genes and controlled by a Lambda cI regulated promoter
part is synthetic. holin and lysozime originates from lambda bacteriophage. the homology regions were taken from E.coli BL21 genome
repressilator RBS Colicin E2 Immunity protein
Colicin E2 Immunity protein with the repressilator RBS
ptrc* is a modified form of ptrc promoter with the -35 and -10 regions being present in the reverse order and the promoter is flanked by fimE recognition sequence. FimE is an invertase capable of inverting any DNA sequence flanked between its recognition seqence. This fim inversion system would help avoid leaky expression. We expressed a new lysis device, DpnI under the control of fim inversion promoter. DpnI is a restriction enzyme capable of chopping up all methylated DNAs. We propose DpnI as a novel lysis device. DpnC and DpnD are obtained from Streptococcus pneumoniae R6 strain.
One should be careful while expressing Dpn genes. Dpn is supposed to chop up the DNA and cause cell death. Hence, no leaky level of Dpn is needed to recover live cells
A composite part between J23100 and antiholin
Transcriptional terminator consisting of a 64 bp stem-loop.
Built by Josh as an intermediate in screening plasmid construction.
This biosafety device for plants consists on two submodules: a male sterility submodule and an identity preservation submodule. The male sterility submodule consist on a barnase specifically expressed in anthers under the regulation of the TA29 tapetum-specific promoter. As result, pollen from these plants is not fertile. The identity preservation submodule expresses the yellow chromoprotein AmilGFP, so that plants can be visually differentiated from non-transgenic plants.
The assembly was performed by GoldenBraid assembly. The barnase was ampliified by PCR from [http://parts.igem.org/Part:BBa_J72173 Part:BBa_J72173] and a mutation in T105P was introduced.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
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 (<partinfo>I13453</partinfo>) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
Conversion of the iGEM parts registry to SBOL2.1