Coding

Part:BBa_K1742003

Designed by: Ivn Casas Rodrigo   Group: iGEM15_Valencia_UPV   (2015-09-04)

Dronpa 145K

The green fluorescent protein Dronpa derive from a tetrameric parent isolated from a stony coral species (Pectiniidae) that was engineered to a monomeric form using both, rational and random mutagenesis [1]. Dronpa fluorescence is switched on upon illumination with violet light (400 nm) and is again switched off upon illumination with cyan light (500 nm). Dronpa protein is contemplated as a visualization tool, and it has recently been appreciated as an optical control element. Upon the introduction of a K145N substitution, a homotetrameric complex is formed that monomerizes upon illumination with cyan light. Generating fusion constructs of the original form DronpaK and the tetrameric mutant DronpaN, enables the reversible association of intramolecular dimmers with violet light [2]. This optogenetic method has not been proved as a transgene expression system yet.

Valencia UPV team has de novo design and engineered an approach for light-directed transcriptional activation of target genes in plants.

The first part consists of DronpaK , fused to a DNA-binding domain (DBD) that binds its operator site nearby the promoter region of a target gene. The second part includes DronpaN (BBa_1742002) fused to the VP16 domain, which acts as a transcriptional activator of a target gene.

When illumination with violet light is applied, DronpaK and DronpaN (BBa_1742002) form a heterodimer in close proximity with the DNA-binding domain operator. Thus, VP16 recruits RNA polymerase and the gene of interest is transcribed.

800px-BBa_1742002_LacIToggleDronpa.png

Figure 1. Representative scheme of DronpaK and DronpaN light-controlled gene expression system.


Employing the GoldenBraid assembly system, both transcriptional units (DBD fused to DronpaK and DronpaN fused to VP16) were assembled in a single plasmid. The complete constructs were made with three different type of DBDs: LexABD (BBa_K1742010), LacIBD (BBa_K1742011) and Gal4BD (BBa_K1742012).

In order to see if our light-dependant toggle switch was functional in plants, the gene of interest assembled (GoldenBraid assembly) with the chimeric promoter (composed by the DBD operator ‘OpLacIBD’, a minimal promoter ‘miniP35S’ and a 5’-UTR region from TMV) was the Luciferase gene. In order to have a control for the Luciferase assay, we needed another construct already available in the GoldenBraid collection. It included the Renilla and P19 genes.

Unfortunatelly results were not observed due to a lack of activation. In order to discard the possibility of lack of expression, a sample of protoplasts transformed with the entire construction of the blue toggle was observed in a confocal microscope irradiating with wavelengths of 405 nm (Dronpa activation), 488nm (Dronpa deactivation).

800px-Valencia_UPVDronpa_fluorescence.png

Figure 2. Fluorescence of Dronpa protein in N. benthamiana protoplasts.Protoplasts were obtained from Agrobacterium-transfected leaves. Two hours after transformation, protoplast sample was collected and observed in a confocal microscope As it can be observed when applying a 405nm laser, fluorescence inside nucleus is observed, besides some fluorescence appears as time goes by. Nevertheless, after irradiating with 488nm the protoplast has lost the majority of its cytoplasm fluorescence as well as its nucleus fluorescence. Finally, after applying a 405nm laser again nucleus fluorescence is reestablished..


References
1. Ando, R., Mizuno, H., Miyawaki, A (2014). Regulated fast nucleocytoplasmatic shuttiling observed by reversible protein highlighting. Science, 306, 1370-1373
2. Zhou XX., Chung HK., Lam AJ., Lin MZ (2012). Optical control of protein activity by fluorescent protein domains. Science 338: 810-814
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
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/reporter/gfp
//chassis/eukaryote/nbenthamiana
//function/reporter/fluorescence
Parameters
colorGreen