cMOF Evidence Base

Synthesis evidence

Bulk protonic conductivity in a cephalopod structural protein

Ordinario D.D., Phan L., Walkup IV W.G. et al. · Nature Chemistry · 2014 · 596-602

5 structured synthesis routes

Completeness describes how fully the route could be reconstructed from the main article and supporting information.

Complete recipeSource: SI

Route 1: Other

1 · Design and Cloning

SolventsSame expression and purification solvents as wild-type route
AdditivesAlanine codons substituted for all GAT/GAC and GAA/GAG codons; 100 ug mL^-1 carbenicillin during expression
Atmospherestandard E. coli expression
Temperature37
Work-upGene synthesized by GeneArt and cloned into pJExpress414 with N-terminal 6x histidine tag; expressed and purified in the same fashion as wild-type reflectin.
ActivationNo porous-material activation; protein purified and lyophilised.
Show 3 structured reagent records
RoleReagentAmount / concentrationSource
OtherDE->A reflectin A1 mutant geneNot specified1 · Design and Cloning
OtherpJExpress414 plasmid with N-terminal 6x histidine tagNot specified1 · Design and Cloning
OtherBL21(DE3) E. coli cellsNot specified1 · Design and Cloning
Complete recipeSource: SI

Route 2: Other

2 · Design and Cloning

SolventsSame expression and purification solvents as wild-type route
AdditivesEmboss shuffleseq/Needle-designed scrambled sequence; 100 ug mL^-1 carbenicillin during expression
Atmospherestandard E. coli expression
Temperature37
Work-upScrambled sequence selected after 20 shufflings and pairwise alignment; expressed and purified in the same fashion as wild-type reflectin.
ActivationNo porous-material activation; protein purified and lyophilised.
Show 3 structured reagent records
RoleReagentAmount / concentrationSource
OtherRandom reflectin A1 scrambled mutant geneNot specified2 · Design and Cloning
OtherpJExpress414 expression vectorNot specified2 · Design and Cloning
OtherBL21(DE3) E. coli cellsNot specified2 · Design and Cloning
Complete recipeSource: SI

Route 3: Drop Cast

6 · Preparation of Reflectin-modified Electrodes

Metal precursorsgold working electrode
SolventsMilli-Q water
Additives0.3 um and 0.05 um alumina polishing slurries
Atmosphereargon-purged phosphate-buffered saline during CV
Timeovernight solvent evaporation
Oxidant / reductant1 M H2SO4 electrochemical etch for gold cleaning
Work-upGold electrodes polished, rinsed, sonicated, etched in 1 M H2SO4, rinsed, then coated by direct deposition of reflectin solution.
ActivationNone
Show 4 structured reagent records
RoleReagentAmount / concentrationSource
Othergold working electrode2 mm diameter6 · Preparation of Reflectin-modified Electrodes
Additivewet alumina slurry0.3 um and 0.05 um6 · Preparation of Reflectin-modified Electrodes
Acidsulfuric acid1 M6 · Preparation of Reflectin-modified Electrodes
Electrolyte10X BupH phosphate buffered salinepH 7.2, 1 M phosphate, 1.5 M NaCl6 · Preparation of Reflectin-modified Electrodes
Complete recipeSource: Both

Route 4: Drop Cast

601 · Methods

Metal precursors4 nm chromium adhesion layer; 40 nm gold or 40 nm palladium electrode layer
Solventsaqueous reflectin solution; acetone and isopropanol for glass cleaning
Atmosphereambient overnight drying; 5% H2/95% Ar used for PdHx formation when palladium electrodes were used
Timeovernight drying
Substrate orientationSiO2/Si p-type boron-doped <100> wafers for d.c./transistor; glass slides for EIS
Oxidant / reductantH2 gas converts Pd electrodes to PdHx for proton-injecting devices
Work-upSiO2/Si cleaned in piranha solution; glass cleaned by sequential acetone/isopropanol sonication; electrodes evaporated by shadow-mask lithography; films mechanically scribed to rectangles spanning electrode pairs.
ActivationNo activation; films dried in air overnight and humidified during measurements.
Show 8 structured reagent records
RoleReagentAmount / concentrationSource
Otheraqueous reflectin solutionNot specified601 · Methods
Otherchromium adhesion layer4 nm601 · Methods
Othergold electrode layer40 nm601 · Methods
Otherpalladium electrode layer40 nm601 · Methods
Reductanthydrogen in argon5% H2/95% Ar601 · Methods
SolventacetoneNot specified601 · Methods
SolventisopropanolNot specified601 · Methods
Oxidantpiranha solution1:3 hydrogen peroxide to sulfuric acid601 · Methods
Complete recipeSource: SI

Route 5: Other

2 · Expression and Purification

SolventsTerrific Broth medium; denaturing buffer: pH 7.4, 50 mM sodium phosphate, 300 mM NaCl, 6 M guanidine hydrochloride; HPLC Buffer A: 99.9% H2O, 0.1% TFA; Buffer B: 95% acetonitrile, 4.9% H2O, 0.1% TFA
Additives100 ug mL^-1 carbenicillin; 250 mM imidazole elution additive
Atmospherestandard E. coli expression; flash frozen in liquid nitrogen before lyophilisation
Temperature37
Work-upInclusion bodies prepared with BugBuster, solubilised in denaturing buffer, purified by HisPur cobalt IMAC, concentrated, reverse-phase C18 HPLC purified, pooled, flash-frozen, lyophilised.
ActivationNo porous-material activation; protein dried by lyophilisation before device solution preparation.
Scalability contextMain text reports optimised protocol yielded >800 mg pure protein per litre of cell culture.
Show 7 structured reagent records
RoleReagentAmount / concentrationSource
OtherpJExpress414 expression vector containing wild-type reflectin A1 geneNot specified2 · Expression and Purification
OtherBL21(DE3) E. coli cellsNot specified2 · Expression and Purification
SolventOvernight Express Instant Terrific Broth mediaNot specified2 · Expression and Purification
Additivecarbenicillin100 ug mL^-12 · Expression and Purification
Additiveguanidine hydrochloride6 M2 · Expression and Purification
Additiveimidazole250 mM2 · Expression and Purification
Solventacetonitrile/H2O/TFA HPLC gradientNot specified2 · Expression and Purification