Constitutive LuxR plus AHL-inducible LacZ
It produces two proteins, first LuxR and then LacZ. LuxR is expressed constitutively. When AHL quorum sensing molecule is present, it activates LuxR. Then, the resulting AHL-LuxR compound would bind to the LuxPR promoter, producing LacZ. LacZ encodes the alpha fragment of beta galactosidase and, in the presence of X-gal, can produce a blue colored molecule in DH5alpha, DH10B and Top10 bacterial strains.
There are few design considerations concerning BBa_K1584035. While cutting the EcoRI and Xbal sites there is a chance that the promoter might be cut off. Therefore, one would need to be extra careful when cutting the sites. Also, after the LuxR a terminator is always required. If there is no terminator, then the LacZ would not function.
Seung Meen Choi, Brittany Lee, Nu Ri Choi, SOOJI LEE, Hyun Min Park
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
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">
PCR amplified from BBa_J33202 without the RBS.
In strains with lacZ-omega (lacZ N-terminal deletion mutant) like DH5alpha, DH10B and Top10, lacZ-alpha restores the beta-galactosidase activity.
This is a copy of the Part:B0014. Only here the part is cloned in the standard vector pSB1C3 instead of the pSB1AK3.
Double terminator consisting of BBa_B0012 and BBa_B0011
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
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 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.
Conversion of the iGEM parts registry to SBOL2.1