Double phasin and intein separed by a flexible protein domain linker
Phasins with intein downstream (separated by a flexible protein domain linker)
Phasins (head and internal domain work as reported in <partinfo>BBa_K300002</partinfo> and <partinfo>BBa_K300003</partinfo>), while intein is described in <partinfo>BBa_K300004</partinfo>. This is a composite part with two phasins (head and internal domain) and an intein separed by a 10aa flexible protein domain linker, which has the function to improve, enhance and ease the folding of these fusion proteins.
Giacomo Zambianchi, Sara Bisi, Paolo Magni
Oligos with a yeast optimized coding for the peptide GENLYFQSGG have been ordered <br\n> The amino acide sequence corresponds to the interdomaine linker of xxx. ''paper''
This is a 10 amino acides long linker peptide which can be used to join protein domains together in a flexible way. So fusion proteins with variable DNA-binding and activation or repression domains might be assembled. <br\n>
To enable the fusion with other domains in frame the vector of this BioBrick has no base pair in between the restriction side and the BioBrick. Furthermore, this coding sequence does not include a start codon.<br\n> For more information about this issus, see:<br\n> Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." <br\n> DSpace http://hdl.handle.net/1721.1/32535 (2006).
It is identical to <partinfo>BBa_K208001</partinfo>, but it lacks the stop codon in order to support protein fusions. It has been designed as a head domain: The Prefix sequence is 5'-GAATTCGCGGCCGCTTCTAG-3' (RFC10 Prefix) The Suffix sequence is 5'-ACTAGTAGCGGCCGCTGCAG-3' (RFC23 Suffix) For these reasons, a tail domain or an internal domain (compatible with RFC23) are ready to be assembled downstream to create protein fusions. CONSTRUCTION METHOD: <partinfo>BBa_K208001</partinfo> (provided by iGEM HQ in pSB3K3 in RFC23 standard) was PCR-amplified/mutagenized with primers: phaP10F 5'-GCTTCTAGATGATCCTCACCCCGGAACA-3' phaPSR: 5'-GCTACTAGTGGCAGCCGTCGTCTTCTTTG-3' in order to delete the stop codon. The PCR product was ran on a 1% agarose gel, gel-extracted, cut with XbaI-SpeI and ligated with <partinfo>pSB1AK3</partinfo> (previously cut with XbaI-SpeI and dephosphorylated). Positive clones were found through digestion screening/sequencing.
Giacomo Zambianchi, Alessandro Ranieri, Manuel Lupotto, Paolo Magni
It is identical to <partinfo>BBa_K208001</partinfo>, but it lacks the stop codon in order to support protein fusions. It has been designed as a head domain: The Prefix sequence is 5'-GAATTCGCGGCCGCTTCTAG-3' (RFC10 Prefix) The Suffix sequence is 5'-ACTAGTAGCGGCCGCTGCAG-3' (RFC23 Suffix) For these reasons, a tail domain or an internal domain (compatible with RFC23) are ready to be assembled downstream to create protein fusions. '''CONSTRUCTION METHOD:''' <partinfo>BBa_K208001</partinfo> (provided by iGEM HQ in pSB3K3 in RFC23 standard) was PCR-amplified/mutagenized with primers: phaPSF: 5'GCTTCTAGAATGATCCTCACCCCGGAACA-3' phaPSR: 5'-GCTACTAGTGGCAGCCGTCGTCTTCTTTG-3' in order to delete the stop codon. The PCR product was ran on a 1% agarose gel, gel-extracted, cut with XbaI-SpeI and ligated with <partinfo>pSB1A3</partinfo> (previously cut with XbaI-SpeI and dephosphorylated). Positive clones were found through digestion screening/sequencing.
Engineered pH-inducible intein (codon optimized for E. coli) - internal domain
The original sequence comes from M. tuberculosis genomic DNA (RecA gene), but it has been highly engineered (according to [1]) to create a pH-inducible protein domain.
Codon optimized for E. coli. The amino acid sequence comes from [1].
RFC23 scar sequence (Silver-Silver mixed site)
Giacomo Zambianchi, Lorenzo Pasotti, Paolo Magni
Conversion of the iGEM parts registry to SBOL2.1