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	There is a significant increase (p=0.0008 in Student t test) of stickiness when the part BBa_J23119-OmpR234 is introduced. The crystal violet staining visually illustrates this effect.</p><br>		There is a significant increase (p=0.0008 in Student t test) of stickiness when the part BBa_J23119-OmpR234 is introduced. The crystal violet staining visually illustrates this effect.</p><br>
−	Team: TAS_Taipei2017 <br>	+
		+	<h3>Characterization and Improving Function (New Data, 2017)</h3>
		+	<p>
		+	<h3>Team: TAS_Taipei 2017</h3>
	Author: Justin Yang <br>		Author: Justin Yang <br>
−	<br>	+
−	Summary:  <br>	+	<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, a negative control which does not express OmpR234.	+	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.
−	<br> <br>	+	<img src="https://static.igem.org/mediawiki/2017/5/54/Fig_3-15_resize.jpeg" width="600px">
−	<img src="https://static.igem.org/mediawiki/2017/5/54/Fig_3-15_resize.jpeg" width="600px">	+
		+	<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>
		+	<h3>CONGO RED ASSAY</h3>
		+
		+	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.
		+
		+	<br><br>
		+	<img src= "https://static.igem.org/mediawiki/parts/a/ac/Webp.net-resizeimage.jpg"> <br>
		+	<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>
	<br>		<br>
−	<b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;SDS-PAGE results show that BBa_K2229100, BBa_2229200, and BBa_K2229300 overexpress CsgD, OmpR234, or both proteins, respectively.</b> Predicted proteins from the curli operons are listed on the right, and E. coli expressing GFP was used as a positive control.
−	<br> <br>
−	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. When bacteria expressing OmpR234 were plated in petri dishes, biofilms appeared thicker compared to controls.
−	<img src="https://static.igem.org/mediawiki/2017/f/f0/Resized.png">
		+	<img src= "https://static.igem.org/mediawiki/2017/9/9c/Omprplates.png"> <br>
		+	<b> When bacteria expressing OmpR234 (BBa_K2229200) were plated in petri dishes with glass coverslips, biofilms appeared thicker compared to controls (BBa_K342003). </b>
		+	<br>
		+	<br>
		+
		+	<h1>BBa_K2229300 Improves the Function of BBa_K342003</h1>
		+
		+	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).
		+	<br>
		+	<h3>CONGO RED ASSAY</h3>
		+	<img src= "https://static.igem.org/mediawiki/2017/4/49/Seventy_percent.jpeg"> <br>
		+	<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><br>
		+
		+	<br>
		+	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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Latest revision as of 03:35, 1 November 2017

OmpR234 protein, with higher effect on Curli promoter

This part is coding for a mutated version of the response regulator OmpR (J Bacteriol. 1998 180(9):2442-9). This protein will be phosphorylated by the associated-sensor EnvZ. This mutated phosphorylated protein is a better activator of the curli csgD promoter than the wild type OmpR. OmpR234 can activate the cryptic curli operons found in all known K12 strains, and then leads to the formation of thick biofilm on glass and polystyrene.

Usage and Biology

This part can be used to induce a constitutive biofilm producing phenotype in bacteria. OmpR234 is now used by several labs in the world (in USA for example, Cornell: Microbiology. 2011 157:1640-50 or Columbia: Water Res. 2008 42:3066-74) and the original paper describing this mutant has been cited 204 times.

Characterization (New data, 2011)

Microscopy tests

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The pIG16 (BBa_J23119-OmpR234 in pSB1A2) and pIG3 (BBa_J23119 part in pSB1A2) were inserted in a strain constitutively expressing GFP, PHL1414 (MG1655 with a chromosomical insertion of GFP). The strains were respectively named S31 and S30.

In sterile empty plates, we have introduced 10mL of M63G, with 100μL of Amp and 100μL of bacteria from an overnight liquid culture.

Then, we have added 3 or 4 sterile glass coverslips and have let them incubate at 30°C during 23 hours. If the strain produces some curli, it will form biofilm on the glass slides. We have washed the slides carefully to eliminate non-adherent bacteria.

To determine if a biofilm was able to develop, we observed the surface of the contaminated coverslips with a fluorescent microscope. After a thorough visual scanning, several optic fields (at least 3) were photographed. One representative picture is presented here.

The complete protocol is available here

We can see that the strain containing the pIG16 plasmid is more adherent than the strain containing the control plasmid pIG3, and is able to produce thick biofilms on glass.

Conclusion :
The BBa_J23119-OmpR234 part increases the stickiness of a part.

Quantitative adherence tests in plates

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To obtain a quantitative characterization, the plasmids were introduced into the PHL1414 strain. The strain harboring the pIG16 plasmid is noted S31. The control strain with the pIG3 plasmid is noted S30.

The effect of each plasmid on the adherence was then measured in 24-wells plates. The strains were seeded as described here

Both planktonic and adherent bacteria have been first collected in each well and the OD600 measured to estimate the total biomass. Each bar represents the mean value of 6 measures, the corresponding standard deviation is indicated.

Conclusion :
The OD600 is not significantly different for the two strains, which shows there is no significant effect of the plasmids on the growth.

The percentage of adherence was then measured for both strains

The measure of the OD600 of both the biofilm fraction and the planktonic fraction allows to calculate the percentage of adherence of the bacteria. Each bar represents the mean value of 6 measures, the corresponding standard deviation is indicated. The crystal violet staining of the attached bacteria will give us a first idea on the biofilm thickness.

Conclusion :

There is a significant increase (p=0.0008 in Student t test) of stickiness when the part BBa_J23119-OmpR234 is introduced. The crystal violet staining visually illustrates this effect.

Characterization and Improving Function (New Data, 2017)

Team: TAS_Taipei 2017

Author: Justin Yang

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.
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.


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).

BBa_K2229300 Improves the Function of BBa_K342003

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

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