Difference between revisions of "Part:BBa K2229100"
| Line 9: | Line 9: | ||
To test the expression of <b>CsgD</b>, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). A culture transformed with the basic part BBa_K805015 (csgD ORF alone) was used as a negative control. We expected to see CsgD around 25 kDa (Brombacher et al. 2006; Martinez & Stock 1997). <b>Compared to negative control, thicker and darker bands at the expected sizes were observed with both BBa_K2229100 (CsgD overexpression) </b>(figure 3-15; proteins bands are marked by asterisks). | To test the expression of <b>CsgD</b>, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). A culture transformed with the basic part BBa_K805015 (csgD ORF alone) was used as a negative control. We expected to see CsgD around 25 kDa (Brombacher et al. 2006; Martinez & Stock 1997). <b>Compared to negative control, thicker and darker bands at the expected sizes were observed with both BBa_K2229100 (CsgD overexpression) </b>(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 | + | 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). |
| + | |||
| − | |||
<!-- --> | <!-- --> | ||
Revision as of 08:12, 12 October 2017
CsgD Expressing Construct
A CsgD-based construct that employs the strong promoter/strong RBS (K880005) combination to up-regulate expression of CsgD, a transcriptional regulator that activates the synthesis of adhesive curli fimbriae in Escherichia coli, which up-regulates biofilm formation.
Characterization
Expression of CsgD Increases Biofilm Formation
To test the expression of CsgD, we ran SDS-PAGE using transformed and lysed E. coli cultures (figure 3-15). A culture transformed with the basic part BBa_K805015 (csgD ORF alone) was used as a negative control. We expected to see CsgD around 25 kDa (Brombacher et al. 2006; Martinez & Stock 1997). Compared to negative control, thicker and darker bands at the expected sizes were observed with both BBa_K2229100 (CsgD 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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]