Coding

Part:BBa_K2442397

Designed by: Ruiyang He, James Provan   Group: iGEM17_Glasgow   (2017-09-07)
Revision as of 02:51, 2 November 2017 by Jmsprovan (Talk | contribs)


GFP C-term split subunit (GFP2)

[[Image:Glasgow_gfp_gate.png|450px|thumb|center| A schematic representation of a final split-GFP AND-gate]


Design

We designed a PCR reaction to generate a C-terminal GFP2 part from E0040 which would work alongside the existing N-terminal GFP1 part K1789003 made by NUDT_CHINA 2015. Primers were designed to amplify the coding sequence of the 83 aa C-terminal fragment as a biobrick compatible part.

Figure 1: Split GFP C-terminal (GFP2) amplification primers. All sequences represented 5’-3’. Colours denote features: blue text = flanking DNA; yellow highlight = Biobrick prefix (F) or suffix (R); purple = new start codon; unformatted text = annealing section of primer. Underlined sequence denotes an error that is discussed in the results section.


Sequencing of the PCR for the GFP C-terminal part revealed an error – the PCR design had incorporated an incorrect version of the BioBrick prefix.

Figure 2: A Biobrick prefix error in GFP C-term part.</b> Alignment of a sequencing read of the split GFP C-terminal to a reference sequence plasmid map. The bases highlighted in red indicate an insertion has occurred at those positions relative to the reference sequence. The insertion corresponds to the use of the “long” Biobrick prefix, rather than the short version which should precede ATG-starting parts. Alignments performed in ApE.

BioBrick parts beginning with ATG must use a shortened version of the prefix ending TAG, instead of the longer version for non-ATG parts which ends TAGAG. The short version ensures ideal spacing between upstream ribosome binding site parts and the start codon. Unfortunately, this oversight of the primer design was not initially noticed our plasmid map sequence for the C-terminal GFP part also contained the error. A part such as this with the extra two nucleotides before the start codon has the potential to be translated at decreased level from an upstream ribosome binding site.


Usage and Biology

Sequence and Features


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 176


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