Difference between revisions of "Part:BBa K2043009:Design"
Line 8: Line 8:
 
===Design Notes===
 
===Design Notes===
 
<html>
 
<html>
−
bpuI-FBD10 laccase sequence was checked and corrected for potential BpiI, BsaI and BsmBI recognition sites. These sites cannot be present in the coding sequence, because the BpiI, and BsaI enzymes are widely used in phytobrick cloning (http://2016.igem.org/Resources/Plant_Synthetic_Biology/PhytoBricks). This BioBrick is the bpul gene (<a href="https://parts.igem.org/Part:BBa_K2043007">Bba_K2043007</a>) codon optimized for E. coli was improved by addition of fabric binding domain 10 (FBD10) on the 5'-end of the sequence using Golden Gate assembly method.<br>
+
Fabric Binding Domain 10 was fused downstream in the 5' end of the original  bpul CDS <br>
 +
FBD 10 is a positive control obtained from literature. This 7 amino acid sequence was obtained by the phage display technique. They screened for small peptide ligands that selectively bind to the Cellulose nano whiskers.<br>
 +
A linker was introduced in the DNA sequence between FBD10 and the CDS of  bpul<br>
 +
His-tag is added in the C-terminal for protein purification <br>
 +
The sequence was codon optimized for E. coli and restriction sites for the restriction enzymes BpiI, BsaI and BsmBI were avoided.
 +
The sequence was confirmed by sequencing and no mutations were observed.
 +
The construction was done by Golden Gate technique using BsaI as restriction enzyme to obtain the desired cohesive ends.
 +
<br>
 +
 
  
−
FBD 10 was a positive control obtained from literature. This 7 amino acid sequence was obtained by the phage display technique. They screened for small peptide ligands that selectively bind to the Cellulose nano whiskers.
 
 
      
 
      
 
</html>
 
</html>

Revision as of 03:42, 28 October 2016

_ bpuI-FBD10 laccase codon optimized for E.coli


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

Fabric Binding Domain 10 was fused downstream in the 5' end of the original bpul CDS
FBD 10 is a positive control obtained from literature. This 7 amino acid sequence was obtained by the phage display technique. They screened for small peptide ligands that selectively bind to the Cellulose nano whiskers.
A linker was introduced in the DNA sequence between FBD10 and the CDS of bpul
His-tag is added in the C-terminal for protein purification
The sequence was codon optimized for E. coli and restriction sites for the restriction enzymes BpiI, BsaI and BsmBI were avoided. The sequence was confirmed by sequencing and no mutations were observed. The construction was done by Golden Gate technique using BsaI as restriction enzyme to obtain the desired cohesive ends.


Source

Bacilus pumillus

References

"Screening for cellulose nanowhiskers binding peptides by phage display." Guo, Jing, et al. 2010 Pittsburgh, Pennsylvania, June 20-June 23, 2010. American Society of Agricultural and Biological Engineers, 2010.