Difference between revisions of "Part:BBa K2229100"
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===Characterization===
 
===Characterization===
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<b>Expression of CsgD Increases Biofilm Formation</b>
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<h3>SDS-PAGE</h3>
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BBa_K2229100 contains and expresses CsgD (BBa_K805015). SDS-PAGE results show CsgD protein around 25 kDa, which matches the expected size.
 +
https://static.igem.org/mediawiki/2017/5/54/Fig_3-15_resize.jpeg
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<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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<h3>CONGO RED ASSAY</h3>
  
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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).
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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 CsgD (BBa_K2229100) in our experiments doubles biofilm production compared to the control BBa_K805015. When bacteria expressing CsgD were plated in petri dishes, biofilms appeared thicker compared to controls.  
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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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https://static.igem.org/mediawiki/2017/2/2e/3-16_resize_resize_resize.jpeg
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<br><br>
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<b>Overexpression of CsgD (BBa_K2229100) doubles biofilm production. 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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<br> <br>
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<h1>BBa_K2229300 Improves the Function of BBa_K805015</h1> <br>
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<h3>Team: TAS_Taipei 2017</h3>
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Author: Justin Yang <br>
  
 +
Our new composite part <b>BBa_K2229300</b> improves the function of two existing parts: BBa_K342003 (<i>ompR234</i> ORF) and BBa_K805015 (<i>csgD</i> ORF). CsgD and OmpR234 are regulators of two curli operons, which contribute to biofilm formation. When both proteins are overexpressed, we hypothesized that twice the amount of curli monomers should be made and exported to form fibers and biofilm.
 +
<h3>SDS-PAGE</h3>
 +
Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) showed two extra bands at 15 kDa and 30 kDa, which were not observed in cultures expressing either CsgD or OmpR234 alone. 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 (<i>Robinson et al.</i> 2006; <i>Uhlich et al.</i> 2009; <i>Shu et al.</i> 2012). This suggests that, as expected, BBa_K2229300 stimulates the production of all curli proteins (predicted proteins and sizes are labeled in the SDS-PAGE figure above).
 +
<h3>CONGO RED ASSAY</h3>
 +
https://static.igem.org/mediawiki/2017/d/d3/Fig_3-19_resize_2.jpeg <br>
 +
<b> Overexpression of both CsgD and OmpR234 (BBa_K2229300) increases biofilm production the most. A) Congo red assay stains biofilms. BBa_K2229300 increases adhesion to glass surfaces. B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm. </b>
  
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<b>Fig. 3-15</b> https://static.igem.org/mediawiki/2017/5/54/Fig_3-15_resize.jpeg
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When all three expression constructs were compared, we find that overexpression of OmpR234 and CsgD together (BBa_K2229300) increased biofilm production the most. BBa_K2229300 also increased adhesion to glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps.
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+
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<b>Congo Red Assay</b>
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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 3-16, 3-19). If biofilms were present, the solution would appear red, which could be quantified by an absorbance value.
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We find that overexpressing CsgD increases biofilm production, as we hypothesized (figure 3-19). Overexpression of CsgD (BBa_K2229100) doubles biofilm production compared to the negative control BBa_K805015 (figure 3-16).
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<b>Fig. 3-16</b> https://static.igem.org/mediawiki/2017/2/2e/3-16_resize_resize_resize.jpeg
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<b>Fig. 3-19</b> https://static.igem.org/mediawiki/2017/d/d3/Fig_3-19_resize_2.jpeg
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When three constructs (BBa_K2229100, BBa_K2229200, BBa_K2229300) were compared, we find that overexpression of CsgD alone upregulated biofilm production, but not to the same degree as BBa_K2229200 and BBa_K2229300.
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Revision as of 05:20, 28 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

SDS-PAGE

BBa_K2229100 contains and expresses CsgD (BBa_K805015). SDS-PAGE results show CsgD protein around 25 kDa, which matches the expected size. Fig_3-15_resize.jpeg 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 CsgD (BBa_K2229100) in our experiments doubles biofilm production compared to the control BBa_K805015. When bacteria expressing CsgD were plated in petri dishes, biofilms appeared thicker compared to controls. 3-16_resize_resize_resize.jpeg

Overexpression of CsgD (BBa_K2229100) doubles biofilm production. A) Congo red assay stains biofilm (red). B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.

BBa_K2229300 Improves the Function of BBa_K805015


Team: TAS_Taipei 2017

Author: Justin Yang

Our new composite part BBa_K2229300 improves the function of two existing parts: BBa_K342003 (ompR234 ORF) and BBa_K805015 (csgD ORF). CsgD and OmpR234 are regulators of two curli operons, which contribute to biofilm formation. When both proteins are overexpressed, we hypothesized that twice the amount of curli monomers should be made and exported to form fibers and biofilm.

SDS-PAGE

Cultures carrying BBa_K2229300 (CsgD and OmpR234 expression) showed two extra bands at 15 kDa and 30 kDa, which were not observed in cultures expressing either CsgD or OmpR234 alone. 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 the SDS-PAGE figure above).

CONGO RED ASSAY

Fig_3-19_resize_2.jpeg
Overexpression of both CsgD and OmpR234 (BBa_K2229300) increases biofilm production the most. A) Congo red assay stains biofilms. BBa_K2229300 increases adhesion to glass surfaces. B) Stained biofilm is solubilized in ethanol. C) Absorbance is measured at 500 nm.

When all three expression constructs were compared, we find that overexpression of OmpR234 and CsgD together (BBa_K2229300) increased biofilm production the most. BBa_K2229300 also increased adhesion to glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps.


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]