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Biology Articles » Biotechnology » Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations » Figures
Figures
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Figure 1
Schematical overview of the vectors used in this study.
A physical map for relevant restriction endonucleases is given for the
plasmid pJOE4056.1 and the location and orientation of the rhaPBAD promotor, the genes encoding eGFP, ampicillin resistance (bla), rop and addiction modules ccdA and ccdB are indicated by triangle and arrows. The transcription terminator sequence (ter) is derived from the E. coli rrnB operon. (Click image to enlarge) |
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Figure 2
Fluorescence intensity of eGFP in E. coli JM109 expressed from the indicated plasmids.
The cells were induced with 0.2% (w/v) L-rhamose and the fluorescence
intensity was measured. Intensity after 2, 4, 6, 8 and 24 h are shown
in (A) fluorescence per 0.01 OD600 and in (B) fluorescence
per ml culture. Values shown are the averages of three independent
experiments, for the measuring points at 24 h the standard deviation is
indicated. (Click image to enlarge) |
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Figure 3
Comparison of plasmid-DNA amounts in the E. coli strain JM109.
Cells were grown at 37°C in LB with ampicillin for 16 h, plasmid DNA
was isolated by a boiling preparation [37] and the supernatants were
analyzed by electrophoresis on a 0.5% agarose-gel and visualized by
EtBr staining. Lanes 1 and 11: 1 Kb DNA Ladder (Invitrogen). (Click image to enlarge) |
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Figure 4
Plasmid stability after 48 h in liquid culture without antibiotic selection.
Cells were grown at 37°C in LB supplemented with 0.2% (w/v) L-rhamnose
for 24 h, starting from this culture fresh medium was inoculated, and
again incubated for 24 h under the same conditions, which roughly
matches 50 generations. The percentage of cells without plasmid or loss
of fluorescence are shown, the values are the averages of three
independent experiments. (Click image to enlarge) |
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Figure 5
Fluorescent light micrographs of L-rhamnose induced E. coli BL21 Rha- without plasmid (A) and BL21 Rha- with pWA21 (B). The bars represent 1 μm length. (Click image to enlarge) |
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Figure 6
Effect of plasmids, carbohydrates and eGFP expression on cell length of different strains.
(A) The strains were grown for 24 h at 30°C in LB supplemented with
0.2% (w/v) L-rhamnose (+), L-arabinose (A) or D-glucose (G) as
indicated in the row 'induction'. For each culture the lengths of 100
cells were determined, the average value and the standard deviation are
shown. The optical density of the cultures is indicated by a black
diamond above the bar. The number of cells per milliliter of culture
were determined by counting them in a Thoma-chamber. (Click image to enlarge) |
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Figure 7
The influence of D-glucose on the eGFP expression in E. coli JM109 with the plasmids pWA21, pWA73.1 and pWA64.1.
The cells were grown for 2 h at 37°C in LB supplemented with D-glucose
as indicated, then shifted to 30°C and induced with 0.2% (w/v)
L-rhamnose and the fluorescence intensity was measured. Intensity after
2, 4, 6, 8 and 24 h are shown in (A) fluorescence per 0.01 OD600 and
in (B) fluorescence per 1 ml culture. Values shown are the averages of
three independent experiments, for the measuring points at 24 h the
standard deviation is indicated. (Click image to enlarge) |
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