[K235022][K235000] (NAND gate, pAra and pLac input signal control, mCherry output signal)
This BioBrick replicates the function of a NAND logic gate (http://en.wikipedia.org/wiki/NAND_gate). The inputs are a) an inducer for the lactose operon, and b) an inducer for the arabinose operon. The output is the production of the mCherry fluorescent protein (J06504). IPTG in medium? Arabinose as best carbon source? mCherry produced? no no yes no yes yes yes no yes yes yes no This system is sensitive to the cellular production of λcI repressor protein. Use with caution in cell strains with arabinose- or lactose-related sensitivity or insensitivity.
Constructed using three-antibiotic assembly. On plasmid pSB1A3.
This part generates a ribokey (J23066) under the control of the lactose promoter. The key will not be produced except in LacI-deficient strains, or when cultured with lactose or inducers such as IPTG.
Constructed using the three-antibiotic assembly method, is on plasmid pSB1C3
[K235018][K235019] (mCherry generator, pAra-controlled ribokey-mediated signal inversion)
This BioBrick generates the mCherry fluorescent protein (J06504), under the control of the λcI-repressed promoter. When the cell is metabolising an inducer for the arabinose promoter (I0500), the mRNA for the λcI repressor protein (C0051) is produced. If a ribokey (i.e., J23066) is present in addition to an inducer, then production of mCherry will halt. This system is sensitive to the cellular production of λcI repressor protein. Use with caution in cell strains with arabinose-related sensitivity or insensitivity.
Constructed using three-antibiotic assembly. On plasmid pSB1C3.
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.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
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 this regulator. This part is incompatible with environments containing lactose or lactose analogs.
Transcriptional terminator consisting of a 64 bp stem-loop.
[key3c] riboregulator for lock3 variants
This part has the same homology region to J01122 as J23007 except that unlike J23007, J23008 does not contain the hairpin that was derived from J01129. J23008 anneals linearly to the stem of the hairpin in J01122 with perfect base pairing and destroys the hairpin in the process and reveals the RBS on J01122. When J01122 is transcribed the RNA secondary structure is such that the RBS hairpins with sequence upstream preventing the ribosome from binding and translation from occuring thereby locking up RFP further down stream. When J23008 is introduced into the cell in trans, we suspect that it will unlock the RBS for RFP as described above allowing translation to occur.
[key3d] riboregulator for lock3 variants
Variant of key3 (see part J01129 (key3), J23007(key3b), J23008(key3c)). Part is in pJ23006, so there is a Ptet upstream of the XbaI site to drive expression. Nevertheless, J23009 is still a basic part allowing both prefix and suffix insertions.
[K235015][K235005] pLambda-controlled mCherry generator
This BioBrick constitutively generates the mCherry fluorescent protein (J06504) unless the λcI repressor protein (i.e., C0051) is present in the cell.
[K235016][K235003] (ribokey-mediated pAra-controlled lambda repressor generator)
This BioBrick generates mRNA for the λcI repressor protein when the arabinose promoter is induced by arabinose or an analogue. The protein is only translated when a ribokey (i.e., J23066) is present in the cell.
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.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Incompatible with host expressing cI repressor.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
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.
cI repressor from E. coli phage lambda (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator (BBa_R0051).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 BBa_C0051 cI repressor is based on the cI repressor from the Elowitz's repressilator. It has been modified to include a rapid degradation LAA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Double terminator consisting of BBa_B0010 and BBa_B0012
Extension of overlapping oligonucleotides
Conversion of the iGEM parts registry to SBOL2.1