DNA gyrase is the only topoisomerase that introduces negative supercoiling into closed circular plasmid molecules. It is a heterotetramer consists of two GyrA and two GyrB subunits. This expression cassette is regulated by an IPTG inducible promoter and will be expressing both Gyr A and Gyr B subunits . Gyr A subunit will bind to the DNA ( gyrase binding site) and is responsible for catalysing DNA breakage and resealing in order to change the level of supercoiling in DNA. Gyr B subunit provides energy for the reaction in the presence of divalent cations such as magnesium cation.
GyrB has a EcoR1 restriction site at 6th bp of the ORF, and it has been removed site directed mutagenesis. The wild type Gyr A ORF has a Pst 1 restriction site at the 1212th bp position so site directed mutagenesis was carried out to remove the restriction site.
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.
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.
Double terminator consisting of BBa_B0010 and BBa_B0012
This is the wild type Gyr A ORF (2627bp) from E.coli , the GyrA subunit is responsible for catalysing the breakage and resealing of DNA strands . A full functional gyrase consist of two GyrA and two GyrB . Gyr A has important tyr122 residue which is involved in forming the phosphotyrosine linkage with the 5' end of the DNA strand in the active site. The full structure of the gyrase is modelled and can be found in our wiki.
This is the E.coli GyrA ORF with a Pst 1 restriction site at the 1212th bp position. SIte directed mutagenesis should be done to remove the site.
This is the 2414bp of GyrB ORF . GyrB subunit has two important lysine residues at 103 and 110th positions which interact and coordinate the positioning of ATP such that the binding and hydrolysis of the ATP will result in the conformational changes which introduce negative supercoiling into the DNA.
The ORF has a EcoR1 restriction site on the 6th bp position.
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