Part:BBa_K1659201:Design
Dispersin B fused at N-terminal with DsbA 2-19 signal sequence
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 316
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 472
Design Notes and Sources
We obtained the peptide sequence for DspB from Kaplan et al's original publication and converted it into a nucleotide sequence using IDT's Codon Optimizer, optimizing based on E. coli K-12's codon table [1].
The gene sequence for DsbA 2-19 was obtained from BBa_K729004, a part comprising DsbA 2-19 fused to the N-terminal of micrococcal nuclease made by [http://2012.igem.org/Team:University_College_London Team UCL 2012]. The part sequence was not annotated, and as such we ran a [http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome Protein BLAST search] using the part's translated sequence to identify the bases that coded for our desired peptide. The sequence for DsbA 2-19 was adopted as-is, without further codon optimization.
We fused the 2-19 peptide sequence of DsbA to the N-terminus of Dispersin B (BBa_K1659200) sequence without any spacer/linker peptide in between. A Hisx6 tag is added at the C-terminus for ease of protein purification using metal-affinity chromatography.
References
[1] Kaplan, J.B. et al., 2004. Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae Biofilms. Journal of Bacteriology, 186(24), pp.8213–8220.