85
15. Previous examples of circularly permuted SBPs [2, 6] can be
used as a guide to identify potential sites for circular permutation, provided that the proteins share sufficient homology.
Otherwise, select sites in disordered loops of the protein (as
indicated by high B-factors in the crystal structures of related
proteins), and avoid the removal of any residues that contribute
to secondary structure. Multiple sites may need to be tested.
As a further check, the CPred server [41] can be used to evaluate the viability of potential sites for circular permutation.
16. Ensure that no SapI sites (GCTCTTC) are present in the synthetic gene, as these will prevent successful cloning into
pDOTS4 or pDOTS10.
17. Cloning into the pDOTS4 or pDOTS10 plasmids by Golden
Gate assembly results in addition of an N-terminal isoleucine
residue and a C-terminal leucine residue to the cloned gene,
which should be taken into account if the linkers between the
fluorescent proteins and recognition domain are optimized.
18. Competent cells with high transformation efficiency (10
7
–
10
8
cfu per μg DNA) are required for this step. If the transformation efficiency of the competent cells is low, plate the entire
volume of electroporated cells by pelleting the cells (18000 × g
on a benchtop centrifuge for 1 min), discarding the supernatant, resuspending the pellet in 100 μL YenB media, and plating the resulting suspension.
19. If no colonies are observed, the ligation by T4 DNA ligase
may have been unsuccessful; if the colonies only contain empty
vectors, digestion of the template gene by SapI may have been
unsuccessful.
20. We recommend sequencing in two reactions, first using a
T7-specific primer (forward) in combination with a SBPspecific primer (reverse), then using an SBP-specific primer
(forward) with a T7-specific primer (reverse), because the
length of the full construct exceeds the length of most Sanger
sequencing reads.
21. High levels of expression can be achieved using pDOTS4 or
pDOTS10 by incubation of E. coli BL21(DE3) transformants
in auto-induction media at 20 °C for 72 h. Expression can be
monitored during growth by removing a 1 mL aliquot from
the culture, centrifuging the sample to pellet the cells, resuspending the pellet in 1 mL water, and measuring the fluorescence spectrum of the resulting cell suspension. With excitation
at 433 nm, an emission peak at 476 nm corresponding to
ECFP and an emission peak at 525 nm corresponding to
Venus should be apparent. Since maturation of the Venus fluorophore is slower than maturation of the ECFP fluorophore,
the Venus peak may not be observed until after 48 h.
Improving FRET Sensors by Ancestral Gene Resurrection
15. Previous examples of circularly permuted SBPs [2, 6] can be
used as a guide to identify potential sites for circular permutation, provided that the proteins share sufficient homology.
Otherwise, select sites in disordered loops of the protein (as
indicated by high B-factors in the crystal structures of related
proteins), and avoid the removal of any residues that contribute
to secondary structure. Multiple sites may need to be tested.
As a further check, the CPred server [41] can be used to evaluate the viability of potential sites for circular permutation.
16. Ensure that no SapI sites (GCTCTTC) are present in the synthetic gene, as these will prevent successful cloning into
pDOTS4 or pDOTS10.
17. Cloning into the pDOTS4 or pDOTS10 plasmids by Golden
Gate assembly results in addition of an N-terminal isoleucine
residue and a C-terminal leucine residue to the cloned gene,
which should be taken into account if the linkers between the
fluorescent proteins and recognition domain are optimized.
18. Competent cells with high transformation efficiency (10
7
–
10
8
cfu per μg DNA) are required for this step. If the transformation efficiency of the competent cells is low, plate the entire
volume of electroporated cells by pelleting the cells (18000 × g
on a benchtop centrifuge for 1 min), discarding the supernatant, resuspending the pellet in 100 μL YenB media, and plating the resulting suspension.
19. If no colonies are observed, the ligation by T4 DNA ligase
may have been unsuccessful; if the colonies only contain empty
vectors, digestion of the template gene by SapI may have been
unsuccessful.
20. We recommend sequencing in two reactions, first using a
T7-specific primer (forward) in combination with a SBPspecific primer (reverse), then using an SBP-specific primer
(forward) with a T7-specific primer (reverse), because the
length of the full construct exceeds the length of most Sanger
sequencing reads.
21. High levels of expression can be achieved using pDOTS4 or
pDOTS10 by incubation of E. coli BL21(DE3) transformants
in auto-induction media at 20 °C for 72 h. Expression can be
monitored during growth by removing a 1 mL aliquot from
the culture, centrifuging the sample to pellet the cells, resuspending the pellet in 1 mL water, and measuring the fluorescence spectrum of the resulting cell suspension. With excitation
at 433 nm, an emission peak at 476 nm corresponding to
ECFP and an emission peak at 525 nm corresponding to
Venus should be apparent. Since maturation of the Venus fluorophore is slower than maturation of the ECFP fluorophore,
the Venus peak may not be observed until after 48 h.
Improving FRET Sensors by Ancestral Gene Resurrection