Intelligent arabinose biosensor with differential response
The parts used to build this device came from several sources. The proteins C and Mor, as well as the Pm and Pmom promoters come from slight modifications of the reported genome of the Mu bacteriophage. The pBad family of promoters comes from British Columbia's 2009 project and are modifications of the wt PBad promoter from E. coli. The other activator and promoter come from Cambridge's 2007 project while the fluorescent proteins and terminators come from the registry of parts.
This part was the final construct of the Tec-Monterrey iGEM Team in 2010. It senses the concentration of arabinose through the PBad family of promoters. Then amplifies it through sensitivity tuners, the activator proteins of the tuners are jammed by reverse arabinose promoters. The sensitivity tuners activate the production of the three fluorescent proteins. This device was created as a proof of concept for the combination of the jammers and sensitivity tuners, rather than using the whole part, the construct design is what should be regarded.
There were no special considerations. This sequence is a proof of concept of the combination of the jammer and sensitivity tuner devices.
Double terminator consisting of BBa_B0012 and BBa_B0011
Synthetized from the reported sequence of Mor protein from Mu bacteriophage. It was optimized for its expression in E. coli.
Mor is a protein of the Mu bacteriophage. It is typically transcribed by the early promoter of the phage and it activates the middle promoter Pm. Since it is the activator of Pm it can be used alongside this promoter to build a sensitivity tuner that increases the POPS output of a construct. The part includes the ribosome binding site, so it can be used directly after a promoter in a genetic construct.
We did an E. coli codon optimization of the sequence and added a ribosome binding site to the sequence before synthesis.
Synthetized from the reported sequence of Pm promoter from Mu bacteriophage.
Pm is the middle promoter of the Mu bacteriophage. It is in charge of the transcription of the activator protein C. Pm is activated by a product of the early transcription, protein Mor. It can be used to build a sensitivity tuner alongside this protein, this construct can be used to increase the amount of POPS in a certain genetic construction.
Synthetized from the reported sequence of Pmom promoter from Mu bacteriophage
Pmom is one of the four late promoters of the Mu bacteriophage. It can be activated by the C protein, which is produced by the middle promoter Pm. Pmom works because of a special configuration of the DNA. It can be used to create a sensitivity tuner alongside the C protein of the same phage. This construction can be used to increase the POPS output of a promoter.
The design of this part had no special considerations
Synthetized from the reported sequence of C protein from Mu bacteriophage. It was optimized for its expression in E. coli.
C is a protein of the Mu bacteriophage. It is typically transcribed by the middle promoter of the phage (Pm) and it activates the four late promoters Plys, Pi, Pp and Pmom. Since it is the activator of the late it can be used alongside them to build a sensitivity tuner that increases the POPS output of a construct. The part includes the ribosome binding site, so it can be used directly after a promoter in a genetic construct.
The sequence was optimized for its production on E. coli and the ribosome binding site was added at the beginning of the sequence.
It was synthesized from the sequence created by British Columbia team in 2009
PBad Weak Rev is the reverse complement of the Arabinose inducible promoter created by British Columbia in 2009. It is a mutation of the wild type PBad promoter that increases the amount of arabinose needed to induce the transcription of the downstream sequences.
We only had to create the reverse complement of the sequence that was already on the registry
This is the pBAD promoter (BBa_I13453) in the opposite orientation. It can be used to drive transcription in the direction of suffix to prefix.
The source of the DNA is the P2 phage genome.
This is the PO promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PO promoter.
mCherry, bacterial with RBS and forward terminator
Combines BBa_B0015 forward terminator with BBa_J06602 mCherry, bacterial with RBS
Site-directed mutagenesis on <partinfo>I13453</partinfo> with the following primers: Forward: TAATCTTATGGATAAAAAAGCTATGGCATAGC Reverse: GCGGATCCTACCTGACGCTTTTTATC
Weaker version of wild type pBAD (<partinfo>I13453</partinfo>).
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type (<partinfo>I13453</partinfo>) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
plasmid DNA supplied by Prof. Richard Calendar, University of California.
The delta activator taken from phiR73 phage acts on a class of inducible promoters (parts I746360 to I746365), inducing their activity to varying degrees. The part sequence does already contain a ribosome binding site (B0034)!
The part does contain a RBS (B0034) already.
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.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
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.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
enhanced yellow fluorescent protein derived from A. victoria GFP
Modificaitons to Clontech EYFP by Reshma Shetty
Transcriptional terminator consisting of a 64 bp stem-loop.
mCherry from Roger Y. Tsien's lab, altered to be BioBrick compatible
mRFP (DsRed) derived, altered to be a biobrick by removing a PstI site and adding in the ends.
Made a point mutation to eliminate a PstI site in the middle and then added BioBrick ends using PCR. Sequenced using primers that bind to the pSB1A2 plasmid on either side of the brick.
Conversion of the iGEM parts registry to SBOL2.1