araC-Pbad regulated XylE - catechol 2,3-dioxygenase from P.putida with terminator
Valentina Herbring, Sebastian Palluk, Andreas Schmidt
XylE - catechol 2,3-dioxygenase from P.putida with terminator
XylE was obtained from the registry <bbpart>BBa_K118021</bbpart>, the terminator is <bbpart>BBa_B0014</bbpart>
Catechol or catechol 2,3-dioxygenases + O(2) is converted by a ring cleavage into 2-hydroxymuconate semialdehyde which is the toxic and bright yellow-coloured substrate. This is a key enzyme in many (soil) bacterial species used for the degradation of aromatic compounds. The enzyme used here itself originating from Pseudomonas putida is a homotetramer of C230 monomers. The tetramerization interactions position a ferrous ion critical for enzymatic activity. It has been deduced that intersubunit interaction is essential to produce a functioning enzyme after performing N and C terminal modifications on the monomer. Coming together the subunits generate an active site. The reaction itself takes place within seconds after the addition by Pasteur pipette or spraying of catechol at a 100mM stock solution diluted with DDH20 (used by our lab.) The toxic byproduct is thought to interfere with cell wall integrity and cellular machinery such that exposed cells gradually die.
The parts were put together using standard assembly [http://partsregistry.org/Assembly:Standard_assembly]
araC-Pbad - Arabinose inducible regulatory promoter/repressor unit
Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121-30 Khlebnikov A, Skaug T, Keasling JD, modulation of gene expression from the arabinose-inducible araBAD promoter,J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.
This promoter can be induced by arabinose. In presens of arabinose the promotor is active.
Double terminator consisting of BBa_B0012 and BBa_B0011
The template DNA was kindly supplied by Dr. Peter Williams of the University of Wales, Bangor. The primer design was based on Genbank sequence M64747 (GI:151718). The sequence reported here was confirmed by sequencing the Biobrick construct.
This part includes the xylE gene from the Pseudomonas putida TOL (naphthalene and xylene degradadative plasmid) pWW0. This gene encodes the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This is a useful reporter gene; colonies or broths expressing active XylE, in the presence of oxygen, will rapidly convert catechol, a cheap colourless substrate, to a bright yellow compound with an absorbance maximum around 377 nm. The part includes the native ribosome binding site, so simply needs to be added after a suitable promoter to act as a reporter. I have previously used this gene to generate whole cell biosensors for various heavy metals. Note that, unlike Xgal etc., catechol in solution is prone to spontaneous oxidation resulting in brown melanin-like polymeric products, so is not stable enough to incorporate into plates or growth media; it should be dripped onto colonies (I use 10 mM catechol in water for this) or added to liquid cultures at a final concentration of about 0.5 mM prior to assay. Note also that there is a SacI site at the very start of the part, when the prefix is included, so this can be used as a replacement vector to introduce PCR products with SacI-SpeI ends into pSB1A2 giving them full Biobrick prefixes and suffixes (but don't forget the G base before the SpeI site). This results in shorter non-complementary tails on PCR primers than using a full prefix or suffix. You can test for colonies that have lost xylE using catechol as described above.
Note that this sequence includes the native ribsomome binding site. Also, when the prefix is included, there is a SacI site at the start, which allows this part to be used as a vector for insertion of PCR products with SacI-SpeI ends into pSB1A2, replacing xylE, giving the full Biobrick prefixes and suffixes (but don't forget the G base before the SpeI site). This results in shorter non-complementary tails on PCR primers than using a full prefix or suffix. You can test for colonies that have lost xylE using catechol as described above.
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.
Conversion of the iGEM parts registry to SBOL2.1