Part:BBa_K1487026

Promoter + Guide RNA (gRNA) target for ctxB

This part contains guide RNA (gRNA) that can target cholera toxin B (ctxB) gene under strong constitutive promoter J23119. This part contains a 42bp binding handle, 20bp targeting region, and a 40bp terminator. This part is ready to use and can form a complex with Cas9 protein to direct a double strand break at a complementary sequence.

This part was tested in Escherichia coli using Reporter part BBa_K1487068, which contains a complementary sequence that is able to be targeted by this gRNA. The reporter is designed with a constitutive promoter so that it continuously produces eYFP (eYFP sequence was cloned from BBa_E0030). Once targeted and cut by the Cas9/gRNA complex, the cells stop producing eYFP and relative fluorescence will decrease. This reporter also confers chloramphenicol resistance, so if it is cleaved in the presence of chloramphenicol-containing media, it will result in cell death. This allowed us to measure the effectiveness of the gRNA/Cas9 targeting by measuring fluorescence as well as cell viability.

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 fluoescence 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). a) Flow cytometry of Escherichia coli 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]