Difference between revisions of "Part:BBa K2229100"
 
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<partinfo>BBa_K2229100 short</partinfo>
 
<partinfo>BBa_K2229100 short</partinfo>
  
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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.  
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A CsgD-based construct that employs the strong promoter/strong RBS (K880005) combination to upregulate expression of CsgD, a transcriptional regulator that activates the synthesis of curli fibers and biofilm formation in <i> Escherichia coli. </i>
  
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===Characterization===
 
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<b>Expression of CsgD Increases Biofilm Formation</b>
 
  
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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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<h1> Construct Design</h1>
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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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This construct was built to upregulate curli production by overexpressing CsgD. We acquired all parts from the iGEM distribution kit: a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, strong RBS (BBa_B0034), csgD (BBa_K805015), and a double terminator (BBa_B0015) to end transcription.<br>
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https://static.igem.org/mediawiki/2017/f/ff/Webp.net-resizeimage_%2813%29.jpg <br>
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<b>For strong CsgD expression (BBa_K2229100), csgD was inserted behind BBa_K880005 (BBa_S05397), and then before BBa_B0015. </b><br><br><br>
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<h3>PCR Check Gel</h3>
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https://static.igem.org/mediawiki/2017/0/00/Webp.net-resizeimage_%2811%29.jpg<br>
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<b>PCR Check for BBa_K2229100 using the forward and reverse primers VF2 and VR. The expected PCR size of BBa_K2229100 (CsgD full construct) is 1100 bp (orange box).</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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<h1>Characterization</h1>
  
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<b>Congo Red Assay</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.
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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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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 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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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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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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<b>Fig. 3-16</b> https://static.igem.org/mediawiki/2017/e/ef/Fig_3-16_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 all three constructs were compared, we find that overexpression of both OmpR234 and CsgD (BBa_K2229300) increased biofilm production the most (figure 3-19). BBa_K2229300 also increased adhesion to our glass coverslips, and we could see a layer of biofilm which remained attached to the glass surface after the washing steps (figure 3-19, A). CsgD alone upregulated biofilm production but not to the same degree as OmpR234 and BBa_K2229300.
 
  
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<h1>References</h1>
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Reinke, A A, and J E Gestwicki. “Insight into amyloid structure using chemical probes.” Chemical biology & drug design., U.S. National Library of Medicine, June 2011, www.ncbi.nlm.nih.gov/pubmed/21457473.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2229100 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2229100 SequenceAndFeatures</partinfo>

Latest revision as of 07:26, 30 November 2017


CsgD Expressing Construct

A CsgD-based construct that employs the strong promoter/strong RBS (K880005) combination to upregulate expression of CsgD, a transcriptional regulator that activates the synthesis of curli fibers and biofilm formation in Escherichia coli.


Construct Design

This construct was built to upregulate curli production by overexpressing CsgD. We acquired all parts from the iGEM distribution kit: a strong promoter and strong RBS combination (BBa_K880005) to maximize protein production, strong RBS (BBa_B0034), csgD (BBa_K805015), and a double terminator (BBa_B0015) to end transcription.
Webp.net-resizeimage_%2813%29.jpg
For strong CsgD expression (BBa_K2229100), csgD was inserted behind BBa_K880005 (BBa_S05397), and then before BBa_B0015.


PCR Check Gel

Webp.net-resizeimage_%2811%29.jpg
PCR Check for BBa_K2229100 using the forward and reverse primers VF2 and VR. The expected PCR size of BBa_K2229100 (CsgD full construct) is 1100 bp (orange box).


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.


References

Reinke, A A, and J E Gestwicki. “Insight into amyloid structure using chemical probes.” Chemical biology & drug design., U.S. National Library of Medicine, June 2011, www.ncbi.nlm.nih.gov/pubmed/21457473.

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]