176
4. pET28a or other non-ampicillin-based resistance marker
plasmids are recommended because Tem1 is produced by the
ampicillin resistance gene. This can interfere with or contaminate the recombinant sensor. Also ampicillin is a competitive
inhibitor of the Tem1 enzyme, so it should be ensured that the
reaction is free of it.
5. These cells are recommended as Tem1 and BLIP are disulfide
bond containing proteins and need an oxidizing environment
for their formation. SHuffle T7 Competent E. coli are modified to have an oxidizing environment in their cytoplasm, so
disulfide bond containing protein can be expressed therein
[17]. Alternatively, expression in the periplasm or re-folding
from denatured proteins can be considered.
6. Nitrocefin is light sensitive and light exposure should be
minimized.
7. Nitrocefin is known to be turned over in highly reducing or
oxidizing environments [18]. We have found that the presence
of strong reducing agents like DTT or ß- mercaptoethanol in
the storage buffer of some macromolecules can lead to rapid
enzyme-independent turnover of nitrocefin. This should be
tested during the initial optimization. If this is found to be the
case, the relevant buffer should be exchanged before adding to
the reaction.
8. The plate reader we use (Perkin Emler Envision) takes 1–2
reads to stabilize the signal. We have been using read 3 as our
initial measurement point. Also, we typically allow the reaction
to proceed for about 30–60 min to ensure that it is complete.
9. It is highly recommended that a master mix comprising PBS,
the sensor, and the macromolecular binder is set up as this
saves time and allows time for the sensor and macromolecule
to bind each other. We typically set up the reaction on ice to
minimize potential thermal unfolding.
10. It is recommended that in each assay, suitable controls are
included. These can include the protease and the sensor, but
without the sterically interfering macromolecular binder to
confirm that both sensor and protease are functional.
11. Some of the potential inhibitors we tested were colored and
had a substantial OD492 value. This can generally be dealt
with by normalizing the sensor reaction against a control reaction with the potential inhibitor and other components except
the sensor itself, or by subtracting the OD at read 1 from all
subsequent reads.
Hui Chin Goh et al.
4. pET28a or other non-ampicillin-based resistance marker
plasmids are recommended because Tem1 is produced by the
ampicillin resistance gene. This can interfere with or contaminate the recombinant sensor. Also ampicillin is a competitive
inhibitor of the Tem1 enzyme, so it should be ensured that the
reaction is free of it.
5. These cells are recommended as Tem1 and BLIP are disulfide
bond containing proteins and need an oxidizing environment
for their formation. SHuffle T7 Competent E. coli are modified to have an oxidizing environment in their cytoplasm, so
disulfide bond containing protein can be expressed therein
[17]. Alternatively, expression in the periplasm or re-folding
from denatured proteins can be considered.
6. Nitrocefin is light sensitive and light exposure should be
minimized.
7. Nitrocefin is known to be turned over in highly reducing or
oxidizing environments [18]. We have found that the presence
of strong reducing agents like DTT or ß- mercaptoethanol in
the storage buffer of some macromolecules can lead to rapid
enzyme-independent turnover of nitrocefin. This should be
tested during the initial optimization. If this is found to be the
case, the relevant buffer should be exchanged before adding to
the reaction.
8. The plate reader we use (Perkin Emler Envision) takes 1–2
reads to stabilize the signal. We have been using read 3 as our
initial measurement point. Also, we typically allow the reaction
to proceed for about 30–60 min to ensure that it is complete.
9. It is highly recommended that a master mix comprising PBS,
the sensor, and the macromolecular binder is set up as this
saves time and allows time for the sensor and macromolecule
to bind each other. We typically set up the reaction on ice to
minimize potential thermal unfolding.
10. It is recommended that in each assay, suitable controls are
included. These can include the protease and the sensor, but
without the sterically interfering macromolecular binder to
confirm that both sensor and protease are functional.
11. Some of the potential inhibitors we tested were colored and
had a substantial OD492 value. This can generally be dealt
with by normalizing the sensor reaction against a control reaction with the potential inhibitor and other components except
the sensor itself, or by subtracting the OD at read 1 from all
subsequent reads.
Hui Chin Goh et al.