Part:BBa_K323080:Experience

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Applications of BBa_K323080

iGEM 2010 team Slovenia used this part along with 4 others [BBa_K323005, BBa_K323021, BBa_K323029, BBa_K323039] to exhibit FRET reconstitution on a predefined DNA scaffold in vivo by transfecting mammalian HEK293 cell line with these 5 plasmids. In particular this BioBrick is part of a FRET subsystem leading to split mCitrine reassembly. It consists of a C terminal fragment of mCitrine, a linker sequence and Zif268 zinc finger DNA binding domain. Reconstitution of a fluorescent protein was observed under confocal microscope. mCitrine was excited with 514nm Argon laser and emmision was detected in 520 to 580 nm window. Combining this 2-part subsystem with the other one, leading to mCerulean reassembly [BBa_K323021, BBa_K323029], FRET on a DNA program can be observed.

Importance

Composite FRET device proved sequential and ordered binding of synthetic zinc finger domains within a predesigned DNA program sequence submitted under part name BBa_323039. This proof of principle shows that directed clustering of functional domains on a DNA program can be achieved.

Scheme of function

Split mCitrine FRET subsystem.

Photos taken under confocal microscope

Both plasmids carrying PBSII_link_nYFP and cYFP_link_Zif268_link were cotransfected into HEK293 cells along with DNA program.

Further development

Similary one can exploit this concept and maybe even furtherly develop it by changing the DNA binding domain and attaching it to split GFPs submitted under part names: BBa_K323011, BBa_K32012, BBa_323032, BBa_323037, BBa_K323055, BBa_K323072. One can also use this split GFPs to observe protein-protein interactions in vivo.

Special feature

Split GFPs were designed so that they overlap at the rassembly point which has previously been proven to lead to enhanced fluorescent signal and better stability of a reconstituted fluorescent protein.

Additional notes

This part had to be cloned from CMV + pSB1AK8 into pSB1C3 as required for part submission. By doing so this part lost eucaryotic terminator. Original plasmid containing this terminator site has been submitted as well, under part name BBa_K323148.

User Reviews

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