BBa_J92210 is a modular, PoPS in/out tunable band pass filter. The part utilizes the Cambridge 2009 iGEM team's sensitivity tuner, Peking U. 2009 iGEM team's AND gate, and the U. of Pavia 2009 iGEM team's logic inverter. An incoming PoPS signal codes for SupDtRNA, then passes through a sensitivity tuner, which either attenuates or amplifies the signal according to user needs (several well characterized sensitivity tuners exist and are well characterized). The signal is then passed through the TetR logic inverter; if the signal is above the the part's inversion symmetry, the signal is attenuated; else, it is amplified. However, signals with amplitudes close to the inverter's line of symmetry pass through with little change. The signal then codes for the T7ptag before reaching a stop codon. For an output signal to pass through this filter, both the SupDtRNA and T7ptag must exist in large enough concentrations for the amber stop codon suppression to take place. In order to change the band width, you can start by swapping out or cascading tuners both before the inverter and immediately after it. However, if you wish to change the middle band or some other fundamental band characteristic (such as degree of attenuation), the inverter must be swapped out. Luckily, any repressor / constitutively on promoter pair should work.
Careful characterization of the tuner and inverter behavior is required to successfully predict the passing band.
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
T7ptag(T7polymerase with amber mutation)
The T7ptag is standardlized from the plasmid kindly provided by Voigt Lab.
T7ptag is a coding sequence of T7 polymerase with 2 Amber mutations. The transcription of T7ptag gene can only lead to the generation of its mRNA, further translation into T7 RNA polymerase is blocked because of the amber mutation. Only in the presence of the Amber mutation suppressor BBa_K228001 SupD-tRNA, it can be translated into functional T7 polymerase. T7ptag is a component of the AND Gate constructed by the Voigt Lab(UCSF)
SupD-tRNA again is from Voigt Lab(UCSF), We standardlize this part by PCR strategy.
The SupD-tRNA part functions as a tRNA, it suppresses the TAG amber stop codon and translate it into a Ser. There is no need to assembly SupD-tRNA with a rbs, since it is not translated. This part, together with the T7ptag part, make an AND Gate, that is, in the presence of both the 2 parts, T7 polymerase is tranlated into a functional polymerase that transcrips a downstream element constructed after a T7 promoter. Which is the output of the AND Gate.
There is a PstI site in the original sequence, but luckily it is near the 3' end of this part, so we designed a mutation in our primer and mutated the PstI site.
Lorenzo Pasotti, Letizia Diamante, Susanna Schiavi, Paolo Magni
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
plasmid DNA supplied by Prof. Richard Calendar, University of California.
The ogr activator taken from P2 phage acts on a class of inducible promoters (parts I746360 to I746365), inducing their activity to varying degrees. The part sequence does already contain a ribosome binding site (B0034)!
The part does contain a RBS (B0034) already.
The original source is the P2 phage genome. For our work the DNA came from plasmids, kindly supplied to us by Prof. Calendar, University of California (Berkeley): Bacteriophage PSP3 and fR73 Activator Proteins: Analysis of Promoter Specificities; Julien and Calendar, 1996; JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5668???5675
This is the PF promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PF promoter.
no special considerations seemed necessary
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.
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.
Conversion of the iGEM parts registry to SBOL2.1