Part:BBa_K2027011:Design

Melanin-Binding Heptapeptide (4D) with CBD and Flag-Lumio-HisTag

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 25
  

Design Notes

Assembling large chains of repetitive DNA sequences is a difficult process due to the high probability of improper annealing during construction. In 2015, our advisor Kosuke Fujishima published a novel method for repetitive DNA strand assembly using small dsDNA blocks with overlap-based redundancies. [14] We designing small chunks of DNA, each coding for a single deca or heptapeptide, with identical 3' and 5' single stranded six nucleotide overlaps. By phosphorylating the DNA blocks' 5' ends with T4 Polynucleotide Kinase (NEB), and allowing the strands to ligate overnight with T4 DNA Ligase (NEB), we were able to generate up to 30 repeated segments per strand (shown in gel image, see protocol). Elongation is halted when a building block has a deletion, insertion, or substitution on its 5' overhang, which prevents further DNA blocks from annealing to its sticky end. These errors led to much higher concentrations of strands with fewer repeats than our target length, which we defined as around ten repeated binding domains. Although we were able to form repetitive strands that could be visualized in a gel with all four of our peptides, along with a randomized hybrid of 4B4/4D, we ran into difficulties in gel extracting all but 4D and P601G at a high enough concentration and purity for insertion into plasmid backbones via Gibson Assembly Protocol.

Repetitive 4D and P601G strands were assembled into pSB1C3 backbones with an upstream flanking constitutive promoter and strong RBS. Downstream of the genes, a cellulose binding domain (CBD) extracted from the 2016 distribution kit part BBa_K1321357 via PCR was included as a proof of concept membrane binding domain, separated from the melanin binding module by a GS linker to preserve each domain's function. Following the CBD, we added a FLAG tag, Lumio tag, and His tag for protein presence verification, extraction, and purification. Finally, a double terminator was included before the biobrick suffix to insure termination of transcription.

Sequence Verification

Source

Howell, R., Revskaya, E., Pazo, V., Nosanchuk, J., Casadevall, A., & Dadachova, E. (2007). Phage Display Library Derived Peptides that Bind to Human Tumor Melanin as Potential Vehicles for Targeted Radionuclide Therapy of Metastatic Melanoma. Bioconjugate Chem., 18(6), 1739-1748. http://dx.doi.org/10.1021/bc060330u

References

Fujishima, K., Venter, C., Wang, K., Ferreira, R., & Rothschild, L. (2015). An overhang-based DNA block shuffling method for creating a customized random library. Sci. Rep., 5, 9740. http://dx.doi.org/10.1038/srep09740