AND Gate LuxR-rbs(B0034)-T7ptag-AraC-SupD
{{PKU_Beijing2009_AND_GATE_FAMILY}} This is a AND GATE in the Ecoli stain DH5a. Use arabinose to induce the transcription of SupD and use IPTG to induce the transcription of T7ptag. If the SupD tRNA is existent, T7ptag can be translated into functional T7 polymerase, and otherwise the translation of T7ptag will come to stop because of some amber stop codon mutations in the T7ptag. The T7 polymerase will activate a T7 promoter. This gate is only one of nine gates(K228038-K228045), which are different in the RBS of T7ptag. For more information: refer to K228000(T7ptag) and K228001(SupD) referrence: Anderson JC, Voigt CA, Arkin AP (2007) Environmental signal integration by a modular AND gate. Mol Syst Biol 3: 133
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.
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.
Built by joining different standardized biobricks together
This is a branch of the AND GATE built by Peking University 2009 iGEM Team. The addition of homoserine lactone can induce the transcription of T7ptag. However, if the SupD tRNA is absent, T7ptag might not be translated into functional T7 polymerase normally because of some amber stop codon mutations in it. We also have constructed a few similar parts with various RBS, which would make the expression regulation of T7ptag gene more convenient by choosing different parts.
The transcription of SupD-tRNA is regulated by the Pbad promoter(<partinfo>BBa_I13453</partinfo>). Arabinose can activate the transciprtion by binding to AraC regulatory protein.
This part was constructed from registry biobricks.
This is a universal part designed to allow induction by HSL of whatever is assembled downstream of the part.
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.
AraC protein(reversed sequence) and Pbad promoter
This part contains AraC protein generator(BBa_K228008) and Pbad promoter(BBa_I13453). The expression of any coding sequence following the Pbad promoter is repressed constitutively by AraC protein and induced by L-arabinose.
The maximum concentration of L-arabinose we tested is 10^-4 M.
The part is RBS->T7ptag->Terminator. K228002 is added to the RBS BBa_B0034 to construct this part. We also have constructed a few similar parts with various RBS, which would make the expression regulation of T7ptag gene more convenient by choosing different parts.
Because the RBS is only about 10bp, so let the BBa_K228002 be the insert and the backbone with RBS be the vector would be more convenient.
Promoter (luxR & HSL regulated -- lux pR)
Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
Constitutive Promoter, High Transcription
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
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)
This part could be connected to a protein coding sequence so that the expression could be induced by L-arabinose
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.
Conversion of the iGEM parts registry to SBOL2.1