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Team:UT-Dallas/Project/results
From 2014.igem.org
Results
Figure 2
a)Fluorescence microscopy of Escherchia coli cells. Cells were transformed with a plasmid that produces the Streptococcus pyogenes Cas9 protein under the control of the pTet promoter and a constitutive gRNA that targets the Vibrio cholerae gene acfA and a plasmid that produces a yellow fluorescence reporter with a 20 base pair sequence from the Vibrio cholerae gene acfA. The first column is a bright field image, the second column is the yellow fluorescence channel, and the third column is the yellow channel with color.
b) Fluorescence microscopy of Escherchia coli cells. Cells were transformed with a plasmid that produces the Streptococcus pyogenes Cas9 protein under the control of the pTet promoter and a plasmid that produces a yellow fluorescence reporter with a 20 base pair sequence from the Vibrio cholerae gene acfA. This is a control for panel (a), having no gRNA. The first column is a bright field image, the second column is the yellow fluorescence channel, and the third column is the yellow channel with color.
c) Serial dilution of the cells in panels (a) & (b). The cells were dilued down to 1/10,000,000 and plated on carbenicillin and chloramphenicol plates.
d) Graph showing colonies versus dilution factor from the serial dilution in panel (c).
Figure 2
a)Fluorescence microscopy of Escherichia coli cells. Cells were transformed with a plasmid that produces the Streptococcus pyogenes Cas9 protein under the control of the pTet promoter and a constitutive gRNA that targets the Vibrio cholerae gene ctxB and a plasmid that produces a yellow fluorescence reporter with a 20 base pair sequence from the Vibrio cholerae gene ctxB. The first column is a bright field image, the second column is the yellow fluorescence channel, and the third column is the yellow channel with color.
b) Fluorescence microscopy of Escherichia coli cells. Cells were transformed with a plasmid that produces the Streptococcus pyogenes Cas9 protein under the control of the pTet promoter and a plasmid that produces a yellow fluorescence reporter with a 20 base pair sequence from the Vibrio cholerae gene ctxB. This is a control for panel (a), having no gRNA. The first column is a bright field image, the second column is the yellow fluorescence channel, and the third column is the yellow channel with color.
c) Serial dilution of the cells in panels (a) & (b). The cells were diluted down to 1/10,000,000 and plated on carbenicillin and chloramphenicol plates.
d) Graph showing colonies versus dilution factor from the serial dilution in panel (c).
Figure 3
a) Flow cytometry of Escherichia cells. The panel on the left shows bacterial cells transformed with with a plasmid that produces the Streptococcus pyogenes Cas9 protein under the control of the pTet promoter and a plasmid that produces a yellow fluorescence reporter with a 20 base pair sequence from the Vibrio cholerae gene ctxB. Doxycycline is added to induce the production of Cas9. The panel on the right shows bacterial cells transformed with with a plasmid that produces the Streptococcus pyogenes Cas9 protein under the control of the pTet promoter and a constitutive gRNA that targets the Vibrio cholerae gene ctxB and a plasmid that produces a yellow fluorescence reporter with a 20 base pair sequence from the Vibrio cholerae gene ctxB.
b) Histogram showing the two populations in panel (a).
C) Total mean fluorescence intensity of the two bacterial populations.
