Biomedical Engineering Reference
In-Depth Information
Fig.
4
Multigenic constructs for BiFC. (
a
) Assembly of four TUs in one T-DNA comprising the two units for BiFC
and the “monitoring/silencing suppressor module.” (
b
) BglII (
Lane 2
) and BanI (
Lane 3
) digestion of the fi nal
multigenic construct pEGB_A-35s:YFN::FUL:T35s-35s:YFC::SOC1:T35s-35s:Renilla:TNos-35s:P19:Tnos-C.
(
c
) Confocal microscopy expression patterns of the negative (YFN::FUL-YFN:Ros) and the positive (YFN::FUL-
YFN:SOC1) BiFC constructs, this latter at two magnifi cations
assays in
Nicotiana benthamiana
. This special module comprises
two TUs, the reporter
Renilla
[
14
] and the TBSV silencing sup-
pressor p19 [
15
] (in both cases the expression is directed by the
35S promoter and the Nopaline Synthase terminator). This special
module prevents silencing effects and allows monitoring the trans-
formation effi ciency using commercial Renilla assay systems. The
renilla-p19 module was previously assembled in a similar fashion as
it is described in Subheadings
3.2
and
3.3
and was subsequently
added to the stored GB collection. The ability to reuse GB devices
is one of the strengths of GB system.
To assemble together the YFN-TFC module with the “moni-
toring/silencing suppressor” module, one, the following steps are
followed (depicted in Fig.
4
):
