Characterization of Putative Ketosteroid Isomerase Variants

Putative KSI variants were identified by sequence relatedness to known KSI variants. Selection of variants was guided by associating putative KSI sequences with T_Growth by species (Engqvist, 2018 (link)). Seventeen variants were synthesized (GenScript or Twist Biosciences) and cloned (Gibson Assembly Protocol, New England Biolabs or Twist Biosciences) into pET-21(+) vectors. KSI variants were aligned using default parameters of Clustal Omega (Madeira et al., 2019 (link)) and the maximum likelihood tree was constructed using IQ-TREE with default parameters (Hoang et al., 2018 (link); Nguyen et al., 2015 (link)). The constructs were expressed in Escherichia coli BL21(DE3) cells and purified as previously described (Kraut et al., 2010 (link)).

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Stark C., Bautista-Leung T., Siegfried J, & Herschlag D. (2022). Systematic investigation of the link between enzyme catalysis and cold adaptation. eLife, 11, e72884.

Publication 2022

Gibson Assembly Protocol

Cells Escherichia coli Tree Vectors

Corresponding Organization :

Other organizations : Stanford University

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Variable analysis

independent variables

Putative KSI variants

dependent variables

T_Growth (growth temperature) by species

control variables

PET-21(+) vector
Escherichia coli BL21(DE3) cells
Purification protocol as previously described (Kraut et al., 2010)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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