Difference between revisions of "Part:BBa K2255007:Design"

(→‎Design Notes)
 
(2 intermediate revisions by 2 users not shown)
Line 8: Line 8:
 
===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>  
+
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.
  
−
As we remove it with [http://2017.igem.org/Team:Aix-Marseille/M13_Design M13 Design], we must put another one. We choose 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:
  
−
In order to be functional, the signal peptide must be cut down from the rest of the protein. Thus, we must add the cleavage site. Using the logiciel SignalP 4.1, we saw that the cleavage is made between the alanine and the glutamate.
+
<code>
 +
MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYG
 +
DTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQ
 +
GQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDSVATDYGAAIDGFIGDVSGLANGNGA
 +
TGDFAGSNSQMAQVGDGDNSPLMNNFRQYLPSLPQSVECRPFVFSAGKPYEFSIDCDKINLFRGVFAFLLYVATFMYVFSTFANILRNKES
 +
</code>
 +
 
 +
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]) <ref>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</ref>, we saw that the cleavage is made between the alanine and the glutamate.
  
 
[[File:T--Aix-Marseille--M13pIII-Sequencesignal.jpeg|400px|center]]
 
[[File:T--Aix-Marseille--M13pIII-Sequencesignal.jpeg|400px|center]]
  
−
In order to gain flexibility, which will help the enzyme to cleave the signal sequence, we add two glycine and one serine residue which we retrotranslate, with the codon biais of E. coli K12.
+
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 protein, thus we choose to make them Freiburg assembly standard with Rfc25 prefix and sufix. This will be helpful in order to assemble our biobrick.
+
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 ([https://parts.igem.org/Assembly_standard_25 Rfc25]) prefix and suffix. This will be helpful in order to assemble our biobrick.
  
 
===Source===
 
===Source===

Latest revision as of 02:47, 2 November 2017


Signal sequence of p3 from M13 (Rfc25)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

T--Aix-Marseille--M13pIII-Sequencesignal.jpeg

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

  1. ↑ 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).
  2. ↑ 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