Difference between revisions of "Part:BBa K2229200"
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===Characterization===
 
===Characterization===
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Expression of CsgD and OmpR234 Increases Biofilm Formation
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Expression of OmpR234 Increases Biofilm Formation
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To test the expression of CsgD and OmpR234, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). Cultures transformed with the basic parts BBa_K805015 (csgD ORF alone) and BBa_K342003 (ompR234 ORF alone) were used as negative controls. We expected to see CsgD around 25 kDa and OmpR234 around 27 kDa (Brombacher et al. 2006; Martinez & Stock 1997). Compared to negative controls, thicker and darker bands at the expected sizes were observed with both BBa_K2229100 (CsgD overexpression) and BBa_K2229200 (OmpR234 overexpression) (figure 3-15; proteins bands are marked by asterisks).
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To test the expression of <b>OmpR234</b>, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). A Culture transformed with the basic part BBa_K342003 (ompR234 ORF alone) is used as a negative control. We expected to see <b>OmpR234 around 27 kDa</b> (Brombacher et al. 2006; Martinez & Stock 1997). <b>Compared to the negative control, thicker and darker bands at the expected sizes were observed with</b><b>BBa_K2229200 (OmpR234 overexpression)</b> (figure 3-15; proteins bands are marked by asterisks).
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In addition to the bands at 25 and 27 kDa, cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in the negative controls. We looked into the other curli operon genes, and found that CsgG is around 30 kDa, whereas CsgA, B, C, E, and F are all around 15 kDa (Robinson et al. 2006; Uhlich et al. 2009; Shu et al. 2012) . This suggests that, as expected, BBa_K2229300 stimulates the production of all curli proteins (predicted proteins and sizes are labeled in figure 3-15).
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<b>In addition to the bands at 25 and 27 kDa, cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in the negative controls.</b> We looked into the other curli operon genes, and found that CsgG is around 30 kDa, whereas CsgA, B, C, E, and F are all around 15 kDa (Robinson et al. 2006; Uhlich et al. 2009; Shu et al. 2012) . This suggests that, as expected, BBa_K2229300 stimulates the production of all curli proteins (predicted proteins and sizes are labeled in figure 3-15).
 
https://static.igem.org/mediawiki/2017/1/14/T--TAS_Taipei--figure_3-15.jpg
 
https://static.igem.org/mediawiki/2017/1/14/T--TAS_Taipei--figure_3-15.jpg
 
<partinfo>BBa_K2229200 parameters</partinfo>
 
<partinfo>BBa_K2229200 parameters</partinfo>
 
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Revision as of 08:43, 11 October 2017


OmpR234 Expressing Construct

An OmpR234-based construct that employs the strong promoter/strong RBS (K880005) combination to up-regulate expression of OmpR234, which in turn activates the csgD promoter to upregulate production of curli fimbriae in Escherichia Coli.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 200
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Expression of OmpR234 Increases Biofilm Formation To test the expression of OmpR234, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). A Culture transformed with the basic part BBa_K342003 (ompR234 ORF alone) is used as a negative control. We expected to see OmpR234 around 27 kDa (Brombacher et al. 2006; Martinez & Stock 1997). Compared to the negative control, thicker and darker bands at the expected sizes were observed withBBa_K2229200 (OmpR234 overexpression) (figure 3-15; proteins bands are marked by asterisks). In addition to the bands at 25 and 27 kDa, cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) contained two extra bands at 15 kDa and 30 kDa, which were not observed in the negative controls. We looked into the other curli operon genes, and found that CsgG is around 30 kDa, whereas CsgA, B, C, E, and F are all around 15 kDa (Robinson et al. 2006; Uhlich et al. 2009; Shu et al. 2012) . This suggests that, as expected, BBa_K2229300 stimulates the production of all curli proteins (predicted proteins and sizes are labeled in figure 3-15). T--TAS_Taipei--figure_3-15.jpg