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pDOTS4, the native SapI site was first removed by sitedirected mutagenesis. To adapt this plasmid for Golden Gate
assembly [31], the ybeJ coding sequence in pRSET FLIPE600n was removed and replaced with SapI-NotI-SapI restriction sites. The SBP gene can be introduced into the pDOTS4
plasmid using Golden Gate assembly (see Subheadings 3.7
and 3.8); the SBP will be fused to a 6× His-Tag and ECFP at
the N-terminus and to Venus fluorescent protein at the
C-terminus. To generate pDOTS10, a biotin tag followed by
a GSSG linker, synthesized de novo (Epoch Life Science)
based on the sequence from the PinPoint
™
Xa-1 plasmid, was
amplified by PCR and cloned into pDOTS4 between the 6×
His-Tag and ECFP using the BamHI restriction site. The SBP
gene can be introduced into the pDOTS10 plasmid using
Golden Gate assembly (see Subheadings 3.7 and 3.8); the SBP
will be fused to a 6× His-Tag, biotin tag, GSSG linker, and
ECFP at the N- terminus, and to Venus fluorescent protein at
the C- terminus. pDOTS4 and pDOTS10 will be made available via Addgene.
2. Usually, protein sequences are used for ancestral reconstruction rather than nucleotide sequences because they are more
highly conserved and therefore retain more phylogenetic signal over longer evolutionary distances; protein sequences are
under stronger selection than nucleotide sequences because
many nucleotide substitutions are synonymous and have low
fitness costs. However, nucleotide- or codon-based phylogenetic analyses are also possible.
3. Information about sequence-function relationships from crystal structures of the reference SBP or other experimental data
is useful for identifying protein sequences that are likely to
have the same or similar binding specificity. If the structure of
the reference SBP is unknown, it may be possible to predict
binding site residues using the structures of homologous
SBPs; these residues should be conserved when the binding
specificity is conserved.
4. Several strategies can be used to increase the diversity of a
sequence dataset: PSI-BLAST, which performs iterative
BLAST searches, can be used to find sequences more distantly
related to the reference SBP; multiple SBP sequences can be
used as input for the BLAST search (provided that they are
actually homologous); programs such as CD-HIT [32] can be
used to filter redundant sequences out of the dataset; BLAST
searches can be restricted to certain taxonomic groups to
obtain sequences from phylogenetically diverse organisms.
Phylogenetic diversity in the sequence dataset is desirable to
enable reconstruction of more ancient (and more thermostable) ancestral proteins.
Ben E. Clifton et al.
pDOTS4, the native SapI site was first removed by sitedirected mutagenesis. To adapt this plasmid for Golden Gate
assembly [31], the ybeJ coding sequence in pRSET FLIPE600n was removed and replaced with SapI-NotI-SapI restriction sites. The SBP gene can be introduced into the pDOTS4
plasmid using Golden Gate assembly (see Subheadings 3.7
and 3.8); the SBP will be fused to a 6× His-Tag and ECFP at
the N-terminus and to Venus fluorescent protein at the
C-terminus. To generate pDOTS10, a biotin tag followed by
a GSSG linker, synthesized de novo (Epoch Life Science)
based on the sequence from the PinPoint
™
Xa-1 plasmid, was
amplified by PCR and cloned into pDOTS4 between the 6×
His-Tag and ECFP using the BamHI restriction site. The SBP
gene can be introduced into the pDOTS10 plasmid using
Golden Gate assembly (see Subheadings 3.7 and 3.8); the SBP
will be fused to a 6× His-Tag, biotin tag, GSSG linker, and
ECFP at the N- terminus, and to Venus fluorescent protein at
the C- terminus. pDOTS4 and pDOTS10 will be made available via Addgene.
2. Usually, protein sequences are used for ancestral reconstruction rather than nucleotide sequences because they are more
highly conserved and therefore retain more phylogenetic signal over longer evolutionary distances; protein sequences are
under stronger selection than nucleotide sequences because
many nucleotide substitutions are synonymous and have low
fitness costs. However, nucleotide- or codon-based phylogenetic analyses are also possible.
3. Information about sequence-function relationships from crystal structures of the reference SBP or other experimental data
is useful for identifying protein sequences that are likely to
have the same or similar binding specificity. If the structure of
the reference SBP is unknown, it may be possible to predict
binding site residues using the structures of homologous
SBPs; these residues should be conserved when the binding
specificity is conserved.
4. Several strategies can be used to increase the diversity of a
sequence dataset: PSI-BLAST, which performs iterative
BLAST searches, can be used to find sequences more distantly
related to the reference SBP; multiple SBP sequences can be
used as input for the BLAST search (provided that they are
actually homologous); programs such as CD-HIT [32] can be
used to filter redundant sequences out of the dataset; BLAST
searches can be restricted to certain taxonomic groups to
obtain sequences from phylogenetically diverse organisms.
Phylogenetic diversity in the sequence dataset is desirable to
enable reconstruction of more ancient (and more thermostable) ancestral proteins.
Ben E. Clifton et al.