Sequence integrated the luxR gene and pluxR promoter together.
We suppose to conduct the quorum sensing AHL-LuxR system in probiotics and S.mutans, thus we connect the luxR coding gene and the pluxR promoter which aims to be activated by the AHL-LuxR complex.
We slightly alter the former arrangement of the V.fischeri AHL-LuxR quorum sensing system to make the pluxR promoter lie below the luxR gene in order to carry on the whole quorum sensing pathway in an align.
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
constitutive promoter and the coding sequence for luxR protein
BBa_K523013 :constitutive promoter region (igem kit) BBa_C0062 : LuxR gene (igem kit) BBa_B0015 : double terminator (igem kit)
allows comunication between gram positive and gram negative to comunicate with the facilitate of phage infection and LuxR luXI system.
In order to fit our idea that we wish the gram-positive S.mutans can communicate with the gram-negative E.coli, we have to find out a quorum sensing system that could be adapted in the both bacterial system. Thus, we use this common quorum sensing system and slightly modify it. We put the signal producing gene, LuxI, at the downstream of the phage lytic gene so that the quorum sensing signal can be spreaded out as S.mutans breakdown.
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.
Double terminator consisting of BBa_B0010 and BBa_B0012
Truncated sequence from BBa_K523013 that this part refers to a constitutive promoter and a RBS.
This truncated sequence come from primer PCR from part BBa_K523013. Reference: http://parts.igem.org/Part:BBa_K523013
The truncated sequence represent a combination of the constitutive promoter region and a RBS region. This transcriptional starter could be applied to Escherichia coli for circuit construction uses.
In order to let E.coli constitutively express the coding sequence, we search for plenty choices of constituve promoter. Eventually, we chose this truncated sequence of part BBa_K523013 as our upstream promoter and RBS sequence.
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.
Conversion of the iGEM parts registry to SBOL2.1