74
4. PHYLIP version 3.695 (http://evolution.genetics.washington.edu/phylip.html).
5. PAML version 4.9 (http://abacus.gene.ucl.ac.uk/software/
paml.html).
6. PyMOL version 1.8 (https://www.pymol.org).
1. NCBI BLAST and PSI-BLAST (http://blast.ncbi.nlm.nih.
gov/Blast.cgi).
2. UniProt-KB BLAST (http://www.uniprot.org/blast).
3. MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle).
4. PhyML (http://www.atgc-montpellier.fr/phyml).
5. Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2).
6. CPred (http://sarst.life.nthu.edu.tw/CPred).
Prepare all solutions using ultrapure water.
1. Synthetic genes encoding the circularly permuted ancestral
proteins (see Subheading 3.7 for design).
2. Primers to amplify the circularly permuted genes with flanking
SapI sites for Golden Gate assembly (see Subheading 3.7 for
design).
3. High-fidelity DNA polymerase (e.g., Phusion Hot Start II
Polymerase, Thermo Scientific).
4. dNTPs.
5. Thin-walled PCR tubes.
6. PCR thermocycler.
7. PCR purification kit (e.g., Wizard
®
SV Gel and Clean-Up
System from Promega).
8. pDOTS4 or pDOTS10 plasmid [6] (see Note 1).
9. SapI (10 U/μL) (e.g., from New England Biolabs).
10. T4 DNA ligase (400 U/μL) with 10× T4 DNA ligase buffer
(e.g., from New England Biolabs).
11. Electrocompetent E. coli TOP10 cells (Invitrogen).
12. Electroporation cuvettes.
13. Electroporator.
14. YenB media: 7.5 g/L yeast extract, 8 g/L nutrient broth.
15. 5 mL culture tubes.
16. Luria-Bertani (LB) agar plates containing ampicillin (10 g/L
tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar,
100 mg/L ampicillin).
17. Materials for colony PCR and analysis by gel electrophoresis.
18. PCR master mix with loading dye for gel electrophoresis (e.g.,
2× PCR Super Master Mix from Biotool).
2.2 Online Servers
2.3 Cloning
Ben E. Clifton et al.
4. PHYLIP version 3.695 (http://evolution.genetics.washington.edu/phylip.html).
5. PAML version 4.9 (http://abacus.gene.ucl.ac.uk/software/
paml.html).
6. PyMOL version 1.8 (https://www.pymol.org).
1. NCBI BLAST and PSI-BLAST (http://blast.ncbi.nlm.nih.
gov/Blast.cgi).
2. UniProt-KB BLAST (http://www.uniprot.org/blast).
3. MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle).
4. PhyML (http://www.atgc-montpellier.fr/phyml).
5. Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2).
6. CPred (http://sarst.life.nthu.edu.tw/CPred).
Prepare all solutions using ultrapure water.
1. Synthetic genes encoding the circularly permuted ancestral
proteins (see Subheading 3.7 for design).
2. Primers to amplify the circularly permuted genes with flanking
SapI sites for Golden Gate assembly (see Subheading 3.7 for
design).
3. High-fidelity DNA polymerase (e.g., Phusion Hot Start II
Polymerase, Thermo Scientific).
4. dNTPs.
5. Thin-walled PCR tubes.
6. PCR thermocycler.
7. PCR purification kit (e.g., Wizard
®
SV Gel and Clean-Up
System from Promega).
8. pDOTS4 or pDOTS10 plasmid [6] (see Note 1).
9. SapI (10 U/μL) (e.g., from New England Biolabs).
10. T4 DNA ligase (400 U/μL) with 10× T4 DNA ligase buffer
(e.g., from New England Biolabs).
11. Electrocompetent E. coli TOP10 cells (Invitrogen).
12. Electroporation cuvettes.
13. Electroporator.
14. YenB media: 7.5 g/L yeast extract, 8 g/L nutrient broth.
15. 5 mL culture tubes.
16. Luria-Bertani (LB) agar plates containing ampicillin (10 g/L
tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L agar,
100 mg/L ampicillin).
17. Materials for colony PCR and analysis by gel electrophoresis.
18. PCR master mix with loading dye for gel electrophoresis (e.g.,
2× PCR Super Master Mix from Biotool).
2.2 Online Servers
2.3 Cloning
Ben E. Clifton et al.