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Part:BBa_K2560011:Design

Designed by: Tobias Hensel   Group: iGEM18_Marburg   (2018-08-21)
Revision as of 19:34, 16 October 2018 by Henselt (Talk | contribs) (→‎Design Notes)


Phytobrick version of 5'Connector Dummy


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal NotI site found at 7
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

The design of “neutral DNA” started with generating 300 bp of random DNA which do not possess recognition sites of BsaI, BsmBI and any of the enzymes used in Biobrick Assembly, using a self-made Matlab script. These sequences were analyzed with the reverse mode of the R2oDNA designer (Casini et al. 2014). The R2oDNA designer can take a DNA sequence as input and quantifies the extent of secondary structures, repeats and forbidden sequence motifs, such as promoter or RBS motifs. Because the genome sequence of V. natriegens is not included in the tool, a BLAST search was performed manually with the sequences to check for homologies with the genome of V. natriegens. The top hit was considered as a fourth score to quantify the quality of a spacer sequence. At the end of this process, four scores were assigned to every sequence, each describing a different characteristic. We decided that spacer sequences with decent scores in each category are better suited as insulators than sequences with a superior score in one and inferior scores in other categories. We developed a Matlab script to find the “best” spacer sequences by increasing quantiles and picking spacers that fall into the lowest quantiles for all four categories. The sequence was synthesized in silico by Daniel Stukenberg and ordered from Integrated DNA Technologies(IDT).

Source

The part was created by annealing single stranded oligonucleotides and subsequent integration into the part entry vector BBa_K2560002 using Golden Gate assembly. If you stuggle with de novo synthesis we recomended this site.

Forward Oligo: CTCGAACAGAATTCGCGGCCGCTTCTAGAGGGAG

Reverse Oligo: CTCACTCCCTCTAGAAGCGGCCGCGAATTCTGTT


References