Composite

Part:BBa_K1618031

Designed by: Leda Coelewij   Group: iGEM15_NRP-UEA-Norwich   (2015-09-09)

Acyltransferase Candidate 3 -Fused to GFP -Expression Construct


K1618030 (P-35s(K1467101)_TP(K1618037):RV3034c(K1618003):GFP(K1467204)_T-35s(BBa_K1618037)) expresses a putative acyltransferase fused to a C-terminal fluorescent reporter in plant cells and targets the gene product to the chloroplast.


The parts were assembled using Golden Gate Assembly and the assembly was tested in a binary plasmid vector backbone prior that can replicated in the shuttle chassis Agrobacterium tumefaciens that transfers the part to plant cells on a T-DNA. For submission to the registry the part was converted to a standard BioBrick by using the Mo-Flipper BBa_K1467400 (complete transcriptional unit flipper) made by the NRP-UEA 2014 team.

Functional characterisation in the model plant Nicotiana benthamiana (Figure 1) indicates that the chloroplast transit peptide is functional and is able to direct the enzyme to the chloroplast where starch is produced.

UEA K1618030 confocal.jpg


UEA 031 confocal .jpg

Figure 1: Constructs BBa_K1618031 contains a fluorescent protein, as well as a chloroplast transit peptide. This is a confocal microscopy image of the construct infiltrated into Nicotiana benthamiana, in which the red structures are the chlorophyll within the chloroplast, and the yellow is the fluorescent fusion protein expressed from the construct.

The results above indicate that our expression construct is functional in confirming the localisation of our part into the chloroplast.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 392
    Illegal BglII site found at 1138
    Illegal BamHI site found at 1843
    Illegal XhoI site found at 1342
    Illegal XhoI site found at 3302
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/eukaryote/nbenthamiana
Parameters
None