Difference between revisions of "Part:BBa K2229100"
 
(22 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2229100 short</partinfo>
 
<partinfo>BBa_K2229100 short</partinfo>
  
−
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.  
+
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>
 +
 
 +
 
 +
<h1> Construct Design</h1>
 +
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>
 +
https://static.igem.org/mediawiki/2017/f/ff/Webp.net-resizeimage_%2813%29.jpg <br>
 +
<b>For strong CsgD expression (BBa_K2229100), csgD was inserted behind BBa_K880005 (BBa_S05397), and then before BBa_B0015. </b><br><br><br>
 +
<h3>PCR Check Gel</h3>
 +
https://static.igem.org/mediawiki/2017/0/00/Webp.net-resizeimage_%2811%29.jpg<br>
 +
<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>
 +
 
 +
 
 +
<h1>Characterization</h1>
  
−
===Characterization===
 
 
<h3>SDS-PAGE</h3>
 
<h3>SDS-PAGE</h3>
 
BBa_K2229100 contains and expresses CsgD (BBa_K805015). SDS-PAGE results show CsgD protein around 25 kDa, which matches the expected size.  
 
BBa_K2229100 contains and expresses CsgD (BBa_K805015). SDS-PAGE results show CsgD protein around 25 kDa, which matches the expected size.  
Line 17: Line 28:
 
<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>
 
<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>
 
<br> <br>
 
<br> <br>
−
<h1>BBa_K2229300 Improves the Function of BBa_K805015</h1> <br>
 
−
<h3>Team: TAS_Taipei 2017</h3>
 
−
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>
 
  
−
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.
+
<h1>References</h1>
 +
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.
  
 
<!-- -->
 
<!-- -->
−
<br>
 
 
<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]