Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein

Figures & data

Figure 1. Plasmid constructs used in this study.

Table 1. Bacterial/plant strains, plasmids and primers used in this study.

	Relevant properties	Source
Strains
E. coli DH5α	Expression host	Lab stock
E. coli TOP10	Expression host	Invitrogen, USA
Agrobacterium LB4404	Used for transgene expression	Invitrogen, USA
N. tabacum L. cv. Petit Havana	Expression host	Lab stock
Plasmids
pBIN	Ori ColE1, Kan^R, P35S, low-copy number	[19]
pCAMBIA1200	Ori pBR322, Cmr^R, P35S, high-copy number	Marker Gene, USA
pQE-30 Xa	Ori ColE1, Amp^R, PT5, low-copy number	Qiagen, USA
Primers
Cellulase qPCR-F	5′-GTGGCGTTTACCACAAACTTAC-3′	This work
Cellulase qPCR-R	5′-GCTGTACTCTTCTCCACCATATAA-3′	This work
CysG qPCR-F	5′-GGCGAAGAGCTGGAAACA-3′	This work
CysG qPCR-R	5′-CGTGAGTGGAATACCCGAATAG-3′	This work
alcR qPCR-F	5′-GAGGAACGCCTGGAATGAA-3′	This work
alcR qPCR-R	5′-CACACTCTGCGTGAGAGAAA-3′	This work
Cellulase-F	5′-AAACACTTCCCTGGTAGGGCAGAA-3′	This work
Cellulase-R	5′-GCGCATTCCAGTTAATCGCAACCT-3′	This work
alcA-F	5′-ACCTAGGATTGGATGCATGCGGAA-3′	This work
alcA-R	5′-ATGTTGGTGAGACTGAGAACCTTGGG-3′	This work
alcR-F	5′-AAACGCCGATCAACCTAGAC-3′	This work
alcR-R	5′-GCTTTGGCAAACGAGTGAATAA-3′	This work

Note: Kanamycin (Kan); Chloramphenicol (Cmr); Ampicillin (Amp); P_35S and P_T5 mean 35S promoter and T5 promoter, respectively.

Table 2. The magnitude of fold differences between all upregulated samples.

		Magnitude of fold-difference
Sample	ΔΔCt	alcA-cel	alcA-cel 1%	alcA-cel 2%	alcA-cel-alcR	alcA-cel-alcR 1%	alcA-cel-alcR 2%	T5-cel
alcA-cel	193.70747	1.00	0.39	0.29	0.03	0.16	0.11	0.30
alcA-cel 1%	501.21319	2.59	1.00	0.76	0.07	0.42	0.27	0.77
alcA-cel 2%	658.91153	3.40	1.31	1.00	0.09	0.55	0.36	1.01
alcA-cel-alcR	7160.69712	36.97	14.29	10.87	1.00	5.95	3.93	10.98
alcA-cel-alcR 1%	1203.10213	6.21	2.40	1.83	0.17	1.00	0.66	1.84
alcA-cel-alcR 2%	1822.73561	9.41	3.64	2.77	0.25	1.52	1.00	2.79
T5-cel	652.38402	3.37	1.30	0.99	0.09	0.54	0.36	1.00

Note: alcA-cel-alcR strain showed the highest expression levels, compared with all other strains. The values are generated by dividing the respective ΔΔCt values (right column over top row). The percentages refer to concentration of ethanol used in the experiment.

Figure 2. Ethanol toxicity assay for E. coli cultures.

Figure 3. Analysis of RT-PCR with gel electrophoresis. Transcription of the three genes (gfp, cellulase and alcR) targeted by correlating detection primers. cDNA (0.3 µL) was used for this test. (A) Transcription of cellulase gene for various strains (1% and 2% indicate ethanol concentration added to the culture). cDNA (0.5 µL) was used for this test (B).

Figure 4. Expression comparison of cellulase genes between different E. coli strains. Clustergram showing all sorted samples, based on the expression intensity (red demonstrate the highest expression level and green the lowest level) (A). Expression variations between different E. coli strains (B).

Figure 5. Expression comparison of alcR gene under different ethanol concentrations.

Figure 6. Epifluorescence micrographs of GFP fluorescence in tobacco leaves expressing p35S-gfp at day three (D–F) and palcA-gfp-alcR at day five (G–I). Fluorescent activity of these lines was compared to wild type leaves (A–C), which showed no fluorescence.

Haseloff J, Siemering KR, Prasher DC, et al. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA. 1997;94(6):2122–2127.

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