pBBknock standard vector for allelic replacement
Synthetic DNA obtained via GenScript synthesis service.
This vector can be specialized to target the desired locus in the Escherichia coli genome and carry out allelic replacement, e.g., knockout, chromosomal integration, mutagenesis, etc. See Casanova et al. (2016), Biol Proced Online for detailed instructions and protocols.
Michela Casanova, Lorenzo Pasotti, Susanna Zucca, Nicolo Politi, Ilaria Massaiu, Cinzia Calvio, Gabriella Cusella, Paolo Magni
This part is the standard BioBricks suffix.
XbaI/SpeI mixed site. Simply used to aid in entry of parts into the registry.
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.
Temperature-sensitive pSC101 replication origin without SpeI site. This part was used to design the pBBknock BioBrick vector for allelic replacement (BBa_J107077).
The origin has been retrieved from pAH123 vector [GenBank: AY048726]. The sequence was extended to the upstream NcoI restriction site and to the downstream stop codon of the ampicillin resistance gene in pAH123. We also modified the origin region by removing the SpeI site, according to the modification present in the available sequence of pSC101 (BBa_I50042), to ensure that the nucleotide changes do not affect the replication origin functioning, e.g., in the widely used pSB4C5 vector. The sequence of pAH123 was obtained via DNA sequencing, since its submitted DNA sequence [GenBank: AY048726] contains errors, e.g., in the temperature-sensitive mutation. The read sequence was then modified to remove SpeI site.
This part is the standard BioBricks prefix.
chloramphenicol resistance cassette (rev)
Reverse complement of <partinfo>BBa_P1004</partinfo> (chloramphenicol resistance cassette without transcriptional terminator).
see <partinfo>BBa_P1004</partinfo> for detailed information.
Lorenzo Pasotti, Manuel Lupotto, Paolo Magni
NsiI restriction enzyme site (PstI compatible overhang)
NsiI recognition site. Digestion with NsiI generates a compatible cohesive end to PstI.
Conversion of the iGEM parts registry to SBOL2.1