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electrophoresis (Fig. 3a) and cell imaging (Fig. 3b), the current
experiment (Fig. 4b, left and Fig. S3, ESI) shows that the same
information can be obtained using a simplistic, wash-free
protocol.Specifically,withprobe1theCSPexpressionlevelscan
be readily analyzed by performing a single incubation step and
by taking a single fluorescence measurement.
DOI: 10.1039/D0CC07095E
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To summarize, a turn-on fluorescent probe (1) integrating a
tri-NTA group and a solvatochromic dye was developed and
used to analyze changes that occur in the expression levels of
His-OmpC in living bacteria. Additional probes that combine
NTA groups with non-responsive fluorescence dyes (2-5) were
also developed and used to confirm the principles underlying
the sensor design. We showed, for example, that although all
probes (1-5) can label His-OmpC, only sensor 1 displayed
fluorescence enhancement upon binding to the engineered
bacteria, indicating the important role the environmentally
sensitive dye plays in the sensor’s response. The contribution of
the tri-NTA unit to the sensor’s labeling efficiency was also
demonstrated by showing that the mono- and bis-NTA probes
(4 and 5) poorly label the bacteria compared with the tri-NTA
probes. Our results also show different applications that can be
achieved with the new tri-NTA probe family developed in this
study (probes 1-3). We have shown that probes 1-3 can be used
to label His-tagged CSPs with different colors; probe 3 can be
used to image them with super resolution, and probe 1 can
straightforwardly analyze changes that occur in their
properties. Probe 1 thus adds a new capabilityto our previous
tri-NTA-basedprobesthatcouldeithersensestructuralchanges
of His-tagged proteins in vitro,5e,for could only label them in
living cells.6 The ability of 1 to determine changes in the
expression level of His-tagged proteins in living bacteria
indicates the potential to expand the analytical toolscurrently
used to monitor changes in protein expression, such as WB or
fluorescence cell imaging. In our future work we aim to test
whether such sensors can be used to quantify protein
expressionlevels inliving cells andwhethertheycanbe applied
to investigate the dynamic properties of cell surface receptors.
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This work was supported by the Israel Science Foundation.
Conflicts of interest
There are no conflicts to declare.
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