Since the TS
didn’t show the expected bistability phenomenon, which indicates that the efficiency of
CRISPRi which targeting the CDS region may be unsatisfactory. We designed three spacers
targeting the CDS regions for these two reporter genes at different locations,
Figure 5.2.1 Binding sites of different spacers in the CDS region of reporter genes
NOR-gate was used to determine the change in expression intensity of different reporter
genes in the presence of only one inducer. After 10h of expression induction with 10ng/ml
aTc or 1% Ara at 37 degrees C, 120 μl of the bacterial solution was taken into a 96-well
plate and measured with an micro mplate reader to obtain its fluorescence intensity.
Interestingly, the spacer targeting the sfGFP CDS region had almost no effect on its
expression intensity, whereas the spacer targeting the mScarlet-I CDS region affected the
expression of sfGFP to some extent in addition to largely reducing the expression intensity
of mScarlet-I (Figure 5.2.2).
suggested that the orthogonality of our spacers targeting mScarlet-I is significantly low
and tends to trigger crosstalk between reporter genes of CRISPRi.
this and the extremely low efficiency of sfGFP CRISPRi as the main reasons why TS didn’t
work. The possible strategies are to target the promoter region to improve the efficiency of
CRISPRi or to use an artificially designed spacer to improve the orthogonality of these two
approach proved to be feasible when we constructed iFFL.
Figure 5.2.2 CRISPRi efficiency of different spacers. The spacers we chose in construction of
the Toggle Switch were mScarlet-Spacer2 and sfGFP-spacer3.