Difference between revisions of "Part:BBa K2442397"
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===Design=== | ===Design=== | ||
− | We designed a PCR reaction to generate a C-terminal GFP2 part from [https://parts.igem.org/Part:BBa_E0040 E0040] which would work alongside the existing N-terminal GFP1 part [https://parts.igem.org/Part:BBa_K1789003 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. | + | We designed a PCR reaction to generate a C-terminal GFP2 coding sequence part from [https://parts.igem.org/Part:BBa_E0040 E0040] which would work alongside the existing N-terminal GFP1 part [https://parts.igem.org/Part:BBa_K1789003 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. |
[[Image:Glasgow_ANDgate_Table_3.png|450px|thumb|center|'''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.]] | [[Image:Glasgow_ANDgate_Table_3.png|450px|thumb|center|'''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.]] |
Revision as of 02:52, 2 November 2017
GFP C-term split subunit (GFP2)
Design
We designed a PCR reaction to generate a C-terminal GFP2 coding sequence 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.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 176