This is a composite part of BBa_F2620, BBa_P1016, BBa_E0840. However, the DNA sequence of the protein coding regions may be changed to facilitate the DNA fabrication process.
This is a measurement plasmid consisting of an AHL-inducible promoter (BBa_F2620), a SpeI site, ccdB, an XbaI site, and a GFP reporter (BBa_E0840). It allows cloning of any device whose input and output are PoPS. The assembled constructs allows tunable control of the input PoPS signal (via AHL) and measurement of the output PoPS signal (via fluorescence).
This measurement plasmid facilitates PoPS->PoPS device characterization. Cloning of a device using XbaI and SpeI into this plasmid allows construction of a composite part that looks identical to a composite part that has been constructed via standard assembly.
Short sequence to promoter addition of 3' A overhang during PCR
<biblio> #Brownstein-Biotechniques-1996 pmid=8780871 </biblio>
This is a short sequence to promoter addition of 3' A overhang during PCR. It should be added to the 5' end of any primers in which the PCR product is going to be TA cloned. It is also a helpful spacer sequence when generating linear DNA fragments to ensure that restriction enzymes will still be able to cut.
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
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
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 tet repressible R0040. Initial characterization work has been done on this device using I13273 as a test device.
Example Data Sheets for F2620
Barry Canton [bcanton@mit.edu] and Anna Labno [labnoa@mit.edu]
This part is derived from BBa_P1010 but it lacks ccdA-.
This part is a differs from BBa_P1011 in that the mutations introduced in BBa_P1011 to remove restriction sites are not present in this part. It does have the "normal" ccd promoter and ccdB coding region. It also has a double TAA stop codon as per BioBrick conventions.
ccdA- was eliminated since it is an inactive form of ccdA and should not be required for the toxic phenotype of ccdB.
This is the sequence of the SpeI/XbaI scar for RBS-CDS junctions in BioBricks standard assembly.
This is a shorter scar to ensure proper spacing between the RBS and CDS.
MfeI restriction enzyme site (EcoRI compatible overhang)
MfeI recognition site. Digestion with MfeI yields a compatible cohesive end to EcoRI.
This is the reverse complement of BBa_G0007.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
NsiI restriction enzyme site (PstI compatible overhang)
NsiI recognition site. Digestion with NsiI generates a compatible cohesive end to PstI.
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
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
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">
Double terminator consisting of BBa_B0010 and BBa_B0012
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