CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

Table 5 Utilization of T7 promoter variants

Plasmid^a	Px^b	Consensus base^c	Naturally occurring promoters^d	Relative promoter strength^e	Standard error
pACYCD-sYFP	consensus			1
pACYCD-sYFP-17A	-17A	T		1.12	0.07
pACYCD-sYFP-17C	-17C			0.92	0.01
pACYCD-sYFP-17G	-17G		φ1.1A, 4C, 4.7	1.54	0.11
pACYCD-sYFP-16C	-16C	A		0.12	0.02
pACYCD-sYFP-16G	-16G			0.48	0.01
pACYCD-sYFP-16T	-16T		φ 4.7	1.54	0.13
pACYCD-sYFP-15C	-15C	A		0.10	0.00
pACYCD-sYFP-15G	-15G			0.16	0.00
pACYCD-sYFP-15T	-15T			1.26	0.09
pACYCD-sYFP-14A	-14A	T		1.15	0.13
pACYCD-sYFP-14C	-14C			0.34	0.02
pACYCD-sYFP-14G	-14G			0.05	0.01
pACYCD-sYFP-13C	-13C	A	φ4c	0.78	0.08
pACYCD-sYFP-13G	-13G			1.26	0.16
pACYCD-sYFP-13T	-13T		φ 3.8, 4.7	1.29	0.04
pACYCD-sYFP-12A	-12A	C		1.50	0.09
pACYCD-sYFP-12G	-12G		φ 3.8	1.78	0.17
pACYCD-sYFP-12T	-12T			1.34	0.13
pACYCD-sYFP-11A	-11A	G	φ OL, 3.8	1.49	0.23
pACYCD-sYFP-11C	-11C			0.06	0.00
pACYCD-sYFP-11T	-11T			0.03	0.00
pACYCD-sYFP-10C	-10C	A		0.09	0.01
pACYCD-sYFP-10G	-10G			0.09	0.01
pACYCD-sYFP-10T	-10T			2.13	0.46
pACYCD-sYFP-9A	-9A	C		0.02	0.00
pACYCD-sYFP-9G	-9G			0.04	0.00
pACYCD-sYFP-9T	-9T			0.02	0.00
pACYCD-sYFP-8A	-8A	T		0.03	0.01
pACYCD-sYFP-8C	-8C			0.03	0.01
pACYCD-sYFP-8G	-8G			0.18	0.01
pACYCD-sYFP-7A	-7A	C		0.02	0.01
pACYCD-sYFP-7G	-7G			0.04	0.01
pACYCD-sYFP-7T	-7T			0.05	0.00
pACYCD-sYFP-6C	-6C	A		0.12	0.02
pACYCD-sYFP-6G	-6G			1.56	0.04
pACYCD-sYFP-6T	-6T			0.53	0.08
pACYCD-sYFP-5A	-5A	C		0.03	0.01
pACYCD-sYFP-5G	-5G			1.47	0.08
pACYCD-sYFP-5T	-5T			0.04	0.01

^aPlasmids in the pACYCD series were constructed in this work
^bPromoter variants are designated as P-nX, where n indicates the position in the promoter and X indicates the base in the non-template strand
^cThe base in the non-template strand of the consensus promoter at the indicated position
^dNaturally occurring T7 RNA Polymerase class II promoters that contain the indicated substitution; all class II promoters have two or more substitutions
^ePlasmid templates were transcribed as described in Fig. 11, and the products (Fluorescent signal) were measured by Synergy™ HTX Multifunctional Microplate Detector
The utilization of each mutant promoter was then expressed relative to that of the consensus promoter when it was present at the position of Px

ISSN: 1754-1611