Difference between revisions of "Part:BBa K2255007:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | The signal sequence is crucial for the excretion of p3 in the periplasm.<ref name="Heilpern">Heilpern, A. J. & Waldor, M. K. pIIICTX, a predicted CTXphi minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae. J. Bacteriol. 185, 1037–1044 (2003).</ref>. As we removed it with [http://2017.igem.org/Team:Aix-Marseille/M13_Design M13 Design], we had to put another one. We chose to use the one coming from M13 as we use ''E. coli'' to produce our phage. | + | The signal sequence is crucial for the excretion of p3 in the periplasm.<ref name="Heilpern">Heilpern, A. J. & Waldor, M. K. pIIICTX, a predicted CTXphi minor coat protein, can expand the host range of coliphage fd to include ''Vibrio cholerae''. J. Bacteriol. 185, 1037–1044 (2003).</ref>. As we removed it with [http://2017.igem.org/Team:Aix-Marseille/M13_Design M13 Design], we had to put another one. We chose to use the one coming from M13 as we use ''E. coli'' to produce our phage. |
This is the complete amino acid sequence of M13 protein 3: | This is the complete amino acid sequence of M13 protein 3: | ||
Latest revision as of 02:47, 2 November 2017
Signal sequence of p3 from M13 (Rfc25)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The signal sequence is crucial for the excretion of p3 in the periplasm.[1]. As we removed it with [http://2017.igem.org/Team:Aix-Marseille/M13_Design M13 Design], we had to put another one. We chose to use the one coming from M13 as we use E. coli to produce our phage.
This is the complete amino acid sequence of M13 protein 3:
MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYG
DTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQ
GQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGA
TGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES
To be functional, the signal peptide must be cut down from the rest of the protein. Thus, we had to add the cleavage site. Using the software ([http://www.cbs.dtu.dk/services/SignalP/ SignalP 4.1]) [2], we saw that the cleavage is made between the alanine and the glutamate.
To gain flexibility, which helps the enzyme to cut the signal sequence, we add two glycines and one serine residue with the codon biais of E. coli K12.
The signal sequence and D1-D2 sequence are designed to make fusion proteins, thus we choose to make them compliant with the Freiburg assembly standard with the (Rfc25) prefix and suffix. This will be helpful in order to assemble our biobrick.
Source
This sequence came from the genome of M13.
References
- ↑ Heilpern, A. J. & Waldor, M. K. pIIICTX, a predicted CTXphi minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae. J. Bacteriol. 185, 1037–1044 (2003).
- ↑ Henrik Nielsen. In Kihara, D (ed): Protein Function Prediction (Methods in Molecular Biology vol. 1611) pp. 59-73, Springer 2017. doi: 10.1007/978-1-4939-7015-5_6 PMID: 28451972