Difference between revisions of "Part:BBa K2229200"
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<h3>SDS-PAGE</h3> | <h3>SDS-PAGE</h3> | ||
BBa_K2229200 contains and expresses OmpR234 (BBa_K342003). SDS-PAGE results show OmpR234 protein around 27 kDa, which matches the expected size (Brombacher et al. 2006; Martinez & Stock 199). This was compared to BBa_K342003, the original part which only contains the ORF. | BBa_K2229200 contains and expresses OmpR234 (BBa_K342003). SDS-PAGE results show OmpR234 protein around 27 kDa, which matches the expected size (Brombacher et al. 2006; Martinez & Stock 199). This was compared to BBa_K342003, the original part which only contains the ORF. | ||
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<br><b>SDS-PAGE results show that BBa_K2229100, BBa_2229200, and BBa_K2229300 overexpress CsgD, OmpR234, or both proteins, respectively. Predicted proteins from the curli operons are listed on the right, and <i>E. coli</i> expressing GFP was used as a positive control.</b><br> | <br><b>SDS-PAGE results show that BBa_K2229100, BBa_2229200, and BBa_K2229300 overexpress CsgD, OmpR234, or both proteins, respectively. Predicted proteins from the curli operons are listed on the right, and <i>E. coli</i> expressing GFP was used as a positive control.</b><br> | ||
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<b> Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.</b> | <b> Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.</b> | ||
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<b> When bacteria expressing OmpR234 (BBa_K2229200) were plated in petri dishes with glass coverslips, biofilms appeared thicker compared to controls (BBa_K342003). </b> | <b> When bacteria expressing OmpR234 (BBa_K2229200) were plated in petri dishes with glass coverslips, biofilms appeared thicker compared to controls (BBa_K342003). </b> | ||
Revision as of 05:17, 28 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
- 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]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 200
- 1000COMPATIBLE WITH RFC[1000]
Characterization
SDS-PAGE
BBa_K2229200 contains and expresses OmpR234 (BBa_K342003). SDS-PAGE results show OmpR234 protein around 27 kDa, which matches the expected size (Brombacher et al. 2006; Martinez & Stock 199). This was compared to BBa_K342003, the original part which only contains the ORF.
SDS-PAGE results show that BBa_K2229100, BBa_2229200, and BBa_K2229300 overexpress CsgD, OmpR234, or both proteins, respectively. Predicted proteins from the curli operons are listed on the right, and E. coli expressing GFP was used as a positive control.
CONGO RED ASSAY
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. If biofilms were present, the solution would appear red, which could be quantified by an absorbance value at 500 nm. Overexpression of OmpR234 (BBa_K2229200) in our experiments lead to about 8 times more biofilm compared to the control BBa_K342003.
Overexpression of OmpR234 (BBa_K2229200) leads to ~8 times more biofilm production than control. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.
When bacteria expressing OmpR234 (BBa_K2229200) were plated in petri dishes with glass coverslips, biofilms appeared thicker compared to controls (BBa_K342003).