Part:BBa_K2229200

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

Protein Gel

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).

Fig. 3-15

Congo Red Assay

After confirming protein expression, we wanted to test if our constructs actually lead to faster and more robust biofilm production. We used Congo Red (CR), a dye commonly used to measure biofilm production (Reinke & Gestwicki 2011). CR solution mixed with bacterial liquid cultures were transferred to 12-well plates with glass coverslips, and incubated at 37˚C for one day. The samples were then washed with PBS and dried. Any stained biofilm on the glass coverslips was solubilized in ethanol, and absorbance was measured at 500 nm (figures 3-17, 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value. We find that overexpressing OmpR234 increases biofilm production as we hypothesized (figure 3-19). Overexpression of OmpR234 (BBa_K2229200) leads to about 8 times more biofilm compared to the negative control BBa_K342003 (figure 3-17 & figure 3-18, A). CGU_Taiwan helped us independently verify our OmpR234 overexpression results using a different dye, crystal violet, which is also commonly used to stain biofilms (figure 3-18, B). Interestingly, both biofilms characterized in our assay are found around the glass coverslip and do not seem to stick well to the glass surface (figures 3-16 & 3-17).

Fig. 3-17

Fig. 3-18

Fig. 3-19