Random libraries were generated
by error-prone PCR (average of 2–3 mutations per clone). Library
1 (phase 1; template = Oluc-N166R) was screened (4,400 variants) with
coelenterazine. Library 2 (phase 2; template = C1A4E) was screened
(4,400 variants) with 11 novel coelenterazine analogues: 3840, 3841,
3842, 3857, 3880, 3881, 3886, 3887, 3889, 3897, and 3900 (Supplementary Figure s4 ). The 11 analogues represented
substitutions at positions 2, 6, and 8 and were considered to be representative
of the entire set of 24 compounds; 2,200 variants were screened with
compounds 3896 and 3894 (Supplementary Figure
s4 ). All hits (improved luminescence) were screened again with
the remaining coelenterazine analogues. Library 3 (phase 3; template
= C1A4E + Q18L/K33N/F54I/F68Y/L72Q/M75K/I90V) was screened in the
context of a mouse Id-X-HaloTag (where X = library) using coelenterazine
and furimazine (Figure1 c). Library screens
were performed on a Freedom robotic workstation (Tecan) as follows:
induced bacterial cultures (in 96-well microtiter plates) were lysed
with a buffer containing 300 mM HEPES pH 8, 200 mM thiourea, 0.3X
Passive Lysis Buffer (PLB, Promega), 0.3 mg mL–1 lysozyme, and 0.002 units of RQ1 DNase (Promega). Assay reagent
containing 1 mM CDTA, 150 mM KCl, 10 mM DTT, 0.5% (v/v) Tergitol,
and 20 μM substrate was then added to equal volumes of lysate.
Samples were measured on a GENios Pro luminometer (Tecan). Secondary
screening to confirm hits (defined as those variants producing greater
luminescence compared to that of the parental clone) and to test combination
sequences was completed using a similar protocol but in manual fashion
and in triplicate.
by error-prone PCR (average of 2–3 mutations per clone). Library
1 (phase 1; template = Oluc-N166R) was screened (4,400 variants) with
coelenterazine. Library 2 (phase 2; template = C1A4E) was screened
(4,400 variants) with 11 novel coelenterazine analogues: 3840, 3841,
3842, 3857, 3880, 3881, 3886, 3887, 3889, 3897, and 3900 (
substitutions at positions 2, 6, and 8 and were considered to be representative
of the entire set of 24 compounds; 2,200 variants were screened with
compounds 3896 and 3894 (
s4
the remaining coelenterazine analogues. Library 3 (phase 3; template
= C1A4E + Q18L/K33N/F54I/F68Y/L72Q/M75K/I90V) was screened in the
context of a mouse Id-X-HaloTag (where X = library) using coelenterazine
and furimazine (Figure
were performed on a Freedom robotic workstation (Tecan) as follows:
induced bacterial cultures (in 96-well microtiter plates) were lysed
with a buffer containing 300 mM HEPES pH 8, 200 mM thiourea, 0.3X
Passive Lysis Buffer (PLB, Promega), 0.3 mg mL–1 lysozyme, and 0.002 units of RQ1 DNase (Promega). Assay reagent
containing 1 mM CDTA, 150 mM KCl, 10 mM DTT, 0.5% (v/v) Tergitol,
and 20 μM substrate was then added to equal volumes of lysate.
Samples were measured on a GENios Pro luminometer (Tecan). Secondary
screening to confirm hits (defined as those variants producing greater
luminescence compared to that of the parental clone) and to test combination
sequences was completed using a similar protocol but in manual fashion
and in triplicate.
