Difference between revisions of "Part:BBa K2229200"
 
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<partinfo>BBa_K2229200 short</partinfo>
 
<partinfo>BBa_K2229200 short</partinfo>
  
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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.
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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 fibers in <i> Escherichia Coli.</i>
  
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<h1> Construct Design</h1>
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===Usage and Biology===
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This construct was built to upregulate curli production by overexpressing OmpR234. 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), ompR234 (BBa_K342003), and a double terminator (BBa_B0015) to end transcription.<br>
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<span class='h3bb'>Sequence and Features</span>
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https://static.igem.org/mediawiki/2017/0/04/Webp.net-resizeimage_%2814%29.jpg <br>
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<partinfo>BBa_K2229200 SequenceAndFeatures</partinfo>
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<b> For strong OmpR234 expression (BBa_K2229200), ompR234 was inserted before BBa_B0015 (BBa_S05398) and then behind BBa_K880005. </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_K2229200 using the forward and reverse primers VF2 and VR. The expected size of BBa_K2229200 (OmpR234 full construct) is 1200 bp (blue box).</b>
  
  
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<h1> Characterization</h1>
  
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===Characterization===
 
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<b>Expression of OmpR234 Increases Biofilm Formation</b>
 
  
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<b>Protein Gel</b>
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<h3>SDS-PAGE</h3>
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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 1997). This was compared to BBa_K342003, the original part which only contains the ORF.
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https://static.igem.org/mediawiki/2017/5/54/Fig_3-15_resize.jpeg
  
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To test the expression of <b>OmpR234</b>, 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 <b>OmpR234 around 27 kDa</b> (Brombacher et al. 2006; Martinez & Stock 1997). <b>Compared to the negative control, thicker and darker bands at the expected sizes were observed with</b><b>BBa_K2229200 (OmpR234 overexpression)</b> (figure 3-15; proteins bands are marked by asterisks).
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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>
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<b>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.</b> 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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<h3>CONGO RED ASSAY</h3>
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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 OmpR234 (BBa_K2229200) in our experiments leads to about 8 times more biofilm compared to the control BBa_K342003.  
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<br><br>
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https://static.igem.org/mediawiki/parts/a/ac/Webp.net-resizeimage.jpg <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>
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<br>
  
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https://static.igem.org/mediawiki/2017/9/9c/Omprplates.png<br>
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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>
  
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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>References</h1>
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<partinfo>BBa_K2229200 parameters</partinfo>
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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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<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 3-17, 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 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).
 
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<b>Fig. 3-17</b> https://static.igem.org/mediawiki/2017/d/d3/3-17_resize_resize_resize_resize.jpeg
 
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<b>Fig. 3-18</b> https://static.igem.org/mediawiki/2017/7/71/318REEEEEEEEEEEESIZE.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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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2229200 SequenceAndFeatures</partinfo>

Latest revision as of 07:27, 30 November 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 fibers in Escherichia Coli.

Construct Design

This construct was built to upregulate curli production by overexpressing OmpR234. 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), ompR234 (BBa_K342003), and a double terminator (BBa_B0015) to end transcription.
Webp.net-resizeimage_%2814%29.jpg
For strong OmpR234 expression (BBa_K2229200), ompR234 was inserted before BBa_B0015 (BBa_S05398) and then behind BBa_K880005.


PCR Check Gel

Webp.net-resizeimage_%2811%29.jpg
PCR Check for BBa_K2229200 using the forward and reverse primers VF2 and VR. The expected size of BBa_K2229200 (OmpR234 full construct) is 1200 bp (blue box).


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 1997). This was compared to BBa_K342003, the original part which only contains the ORF. 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 OmpR234 (BBa_K2229200) in our experiments leads to about 8 times more biofilm compared to the control BBa_K342003.



Webp.net-resizeimage.jpg
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.

Omprplates.png
When bacteria expressing OmpR234 (BBa_K2229200) were plated in petri dishes with glass coverslips, biofilms appeared thicker compared to controls (BBa_K342003).

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 200
  • 1000
    COMPATIBLE WITH RFC[1000]