A device that can accepts the 3--O-C6-HSL and then produces 3-O-C12-HSL and ECFP reporter.
Besides the leuB from E.coli BL21 cloned with PCR by ourselves, all the other parts are from the iGEM, including F2622, K081009, B0034, E0030, B0015.
This composite is a device of function. it is composed of plac(R0011), RBS(B0034), luxR(C0062), double terminator(B0015), plux(R0062), RBS(B0030), lasI(C0078), RBS(B0034), leuB(K594002), RBS(B0034), ECFP(E0030), double terminator(B0015). When IPTG is added, the plac is activated, producing luxR, and then the plux will be activated when 3-O-C6-HSL is added. The 3-O-C6-HSL units with luxR and form a complex that can bind to the lux cassette in plux so that the transcription of lasI, leuB and ECFP happen, producing 3-O-C12-HSL,leuB and the ECFP reporter.
When ligate the RBS with others, 3A assembly is not as efficient as standard assembly.
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.
enhanced yellow fluorescent protein derived from A. victoria GFP
Modificaitons to Clontech EYFP by Reshma Shetty
Double terminator consisting of BBa_B0010 and BBa_B0012
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the lac repressible R0011.
lasI: autoinducer synthetase for PAI from Pseudomonas aeruginosa. Strong RBS (efficiency=0.6).
subunit of a 3-isopropylmalate dehydrogenase from E.coli BL21
This leuB is from the leu operon in BL 21(E.coli),we get BL21 from Takara and found its sequence in NCBI, then cloned it with a Pfu PCR.
LeuB is essential to leucine biosynthesis, so it is a necessary gene for E.coli's well growth. The leu operon is responsible for the leucine biosynthesis, including leuA, leuB, leuC, leuD and leuO. Regulation Summary Diagram: Summary: 3-isopropylmalate dehydrogenase (LeuB) carries out the third step in leucine biosynthesis, catalyzing the conversion of 3-isopropylmalate to 2-isopropyl-3-oxosuccinate. 3-isopropylmalate dehydrogenase oxidizes 3-isopropylmalate with NAD+ to generate 2-isopropyl-3-oxosuccinate and NADH. The kMs listed below were determined at 40?? C, as was the kcat value of 69/s. A crystal structure of 3-isopropylmalate dehydrogenase to 2.1 ?? resolution reveals that it is a dimer.
Besides, when we were looking for leuB gene, we also try to find its RBS, but unluckily, we didn't find its RBS on NCBI, its location is closely linked to leuA's location.
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.
Promoter (lacI regulated, lambda pL hybrid)
represillator of Elowitz and Leibler (2000)
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. 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 increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). 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 Brian Chow
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">
autoinducer synthetase for PAI from Pseudomonas aeruginosa
coding region for lasI protein, which produces the chemical signal AI-1
Conversion of the iGEM parts registry to SBOL2.1