Viton tubing

The assembled YDRH
(Figure 1) was part
of a closed-loop dynamic flow system that recirculated collection
Milli-Q water solvent by a peristaltic pump (Reglo 2-channel Var-Speed
Analog pump from Cole Parmer, Court Vernon Hills, IL) at 100 mL/min
between the inside surface of the test disposable nitrile glove and
the outside surface of the CPC Solvex powderless unsupported/unlined
nitrile glove (Ansell, Iselin, New Jersey) that protected the YDRH.^{10 (link),11 (link)} The inlet and outlet of the YDRH were connected to the sampling
point by Viton tubing (2.79 mm inner diameter extension and three-stop,
Cole Parmer, Vernon Hill, IL). The hand in exposure situations was
immersed inverted, held by the wrist from a clamp, in limonene contained
in a vacuum desiccator (5-L from Fisher Scientific) within a Precision
Econotherm Laboratory Oven (Fisher Scientific) at 35 °C (Figure 1). The hand was powered
as described elsewhere to produce a 1.8 kg clenching force.^{8 (link),10 (link),11 (link)} Thicknesses of the conditioned
and reconditioned gloves after each permeation experiment were measured
with an Electronic Digital Micrometer Model CO-030025 (0–25
mm, 0.001 mm resolution) from Fisher Scientific. Permeation experiments
were performed in triplicate.

T4 DNA ligase and restriction endonucleases were obtained from New England Biolabs. DNA Polymerase Mastermix was from Denville Scientific. Ni-NTA Superflow, QIAprep Miniprep kits, and QIAquick gel extraction kits were purchased from Qiagen. All reagents and cofactors were from Sigma-Aldrich or Thermo Fisher Scientific. Oligonucleotide primers were synthesized by Valuegene and IDT. Gene sequencing and gene synthesis were performed by Genewiz and Twist Bioscience. Assembly master mix (AMM) used for cloning was prepared as outlined in^{19 (link)}. All DNA and protein concentrations were measured with a Thermo Fisher Scientific Nanodrop 1000 Spectrophotometer. Vials for small scale reactions, for gas chromatography and 96-well plates were purchased from VWR. Viton tubing was purchased from Cole-Parmer Instrument Company. Colorimetric enzyme-coupled assays were performed in 96-well plates and measured with Molecular Devices SpectraMax M5 microplate reader. When performing large numbers of assays simultaneously, Integra Viafill and Integra Viaflo instruments were used for reagent additions. Colonies for screening of mutant variants were selected automatically using a Molecular Devices Qpix 420 colony picker.