Difference between revisions of "Part:BBa K1583000"

 
(Complete addition K1583100 text)
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1583000 short</partinfo>
 
<partinfo>BBa_K1583000 short</partinfo>
  
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This gene encodes for the protein CsgA. CsgA is a curlin subunit that can self-assemble into curli after nucleation on CsgB. They are produced in an unpolymerized form and secreted from the cell.
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<html>
  
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CsgA is naturally produced by e.g. E. coli K-12 MG1655 from the operon CsgBA. A second operon, CsgDEFG encodes four additional proteins that are required for the curli assembly and secretion.
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This part is meant to express the <i>csgA</i> gene under control of L-rhamnose-inducible promoter.
 +
 
 +
CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG.
 +
 
 +
This part was designed to be able to control the nanowire formation.
 +
 
 +
<h3>Characterization</h3>
 +
<p>
 +
This part was characterized in three different experiments:
 +
<li> Fluorescence assay </li>
 +
<li> Crystal Violet assay </li>
 +
<li> Transmission electron microscopy </li>
 +
</p>
 +
 
 +
<h4> Fluorescence assay </h4>
 +
<p>
 +
To be able to ensure that CsgA is expressed, we added a gene encoding for GFPmut3 (<a href="https://parts.igem.org/Part:BBa_I13504">BBa_I13504</a>) under induction of the same rhamnose promoter (<a href="https://parts.igem.org/Part:BBa_K1583112">BBa_K1583112</a>) to check that the promoter works. In this experiment, the fluorescence signal of our csgA construct and csgA-GFP (I13504) constructs was recorded in time after induction with no, 0.2% (w/v) or 0.5% (w/v) rhamnose. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell.All conditions were carried out in triplicates to be able to do a statistical analysis on the data. The different experiments were induced in a 96 well plate. The OD600 and fluorescence signal was recorded in a plate reader during a 18 hour period of induction at 30°C.
 +
</p>
 +
 
 +
<p>
 +
In Fig. 1, the fluorescent signal was normalized by the number of cells and plotted as a function of time. The red bars denote the error within each ID.
 +
</p>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png" width="100%" height="100%">
 +
<figcaption>
 +
<b>Fig. 1</b>: Fluorescence signal normalized by the number of cells for 0% (w/v), 0.2% (w/v) and 0.5% (w/v) rhamnose with the csgA and csgA-GFPmut3 construct. The error bars are included for all experiments.
 +
</figcaption>
 +
</figure>
 +
 
 +
<p>
 +
As can be seen from Fig. 1, only the experiments with 0.2% (w/v) and 0.5% (w/v) rhamnose induction with the csgA-GFPmut3 construct gave a clear increase in fluorescence signal in time. All other experiments, gave similar levels of fluorescence, slightly increasing in time. Furthermore, it can be seen that a higher induction level of rhamnose leads to an increase in GFPmut3 and thus fluorescence. Finally, as the fluorescence signal is normalized by the cell density, one can make statements about the activity of the rhamnose promoter. The promoter seems to not be active right after induction, but more after 3 or 4 hours. This is in accordance with data from literature (Wegerer et. al), in which a low amount of fluorescence with a rhamnose promoter was observed after 2 hours of induction.
 +
</p>
 +
 
 +
<p>
 +
With this kinetic experiment, we have proven that the rhamnose promoter does indeed induce the expression of the <i>csgA</i> gene.
 +
</p>
 +
 
 +
<h4> Crystal violet assay </h4>
 +
<p>
 +
The assay above showed that the bacteria that we engineered for the project is capable of producing the CsgA proteins after induction with L-rhamnose. However, this did not yet prove that curli are formed. In order to assess whether our CsgA-producing bacteria can produce these nanowires, our team adapted the protocol from Zhou et al. (2013) that employs crystal violet (methyl violet 10B) for dying the biofilm-making bacteria that attaches to the surface. In the experiment, our CsgA-producing strain of <i>E. coli</i> is induced at a high (0.5% w/v), low (0.2% w/v) and no (0% w/v) concentration of L-rhamnose. Furthermore, <i>csgA</i> deficient bacteria transformed with an empty plasmid (pSB1C3) are used as control.
 +
</p>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/4/46/96_wells_plate_Crystal_Violet_K1583100.jpg" width="50%" height="100%">
 +
<figcaption>
 +
<b>Fig. 3</b>: 96-well plate used to test the curli formation capability of our designed constructs. The first two rows (A-B) contained cultures with 0% w/v of rhamnose. The columns D-E and G-H were inducted with L-rhamnose to a final concentration of 0.2% (w/v) and 0.5% (w/v) respectively. The green boxes show the strains carrying BBa_K1583100, and the red ones the control strain with pSB1C3.
 +
</figcaption>
 +
</figure>
 +
 
 +
<p>
 +
In the end, the wells were diluted with ethanol so all the content can dissolve in the liquid phase. We measured the absorbance at 590 nm of wavelength for all the samples, obtaining the following results (figure 4.).
 +
</p>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/1/1b/Crystal_Violet_result_K1583100.JPG" width="70%" height="100%">
 +
<figcaption>
 +
<b>Fig. 4</b>: Microtiter Plate Assay results for testing curli formation. CTRL0, CTRL2 and CTRL5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells with pSB1C3, used as a control. CSGA0, CSGA2 and CSGA5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells with BBa_K1583000. The termination 0, 2 and 5 denote the induction with no rhamnose (0), 0.2% w/v (2) and 0.5% w/v (5).
 +
</figcaption>
 +
</figure>
 +
 
 +
<p>
 +
The cells induced with l-rhamnose showed an increased absorbance at 590 (the peak of the dye) when compared to the empty-plasmid controls, and also with the non-induced sample. That results clearly demonstrate how our engineered cells with BBa_K1583100 successfully make biofilms, regarding that the empty plasmid controls and the analysed samples have significantly (Table 1.) higher crystal violet retention. On the other hand, a higher concentration of rhamnose is not leading to a higher expression. The cells induced with a 0.2% w/v of rhamnose seem to create better a biofilm structure. However, this could be a consequence of different growth patterns; the cultures induced with 0.5% w/v of rhamnose could have stopped duplicating earlier, so the cell concentration could have decreased. So, the dyed area could be consequently smaller.
 +
</p>
 +
</html>
 +
 
 +
{|style="color:black" cellpadding="4" cellspacing="2" border="1" align="middle"
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! style="background:#0084A7;"|'''Sample'''
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! style="background:#0084A7;"|'''p-value'''
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! style="background:#0084A7;"|'''Significant difference (10%)'''
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|-
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|'''CTRL2 & CSGA2'''
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|0.0426
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|Yes
 +
|-
 +
|-
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|'''CTRL5 & CSGA5'''
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|0.0646
 +
|Yes
 +
|-
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|'''CTRL0 & CTRL2'''
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|0.5034
 +
|No
 +
|-
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|'''CTRL0 & CTRL5'''
 +
|0.9113
 +
|No
 +
|}
 +
 
 +
 
 +
<html>
 +
<!--Table formatting from https://parts.igem.org/Part:BBa_K1150020 -->
 +
<p>
 +
With the experiments described previously we achieved nanowire formation with strains containing the BBa_K1583100 biobrick.
 +
</p>
 +
 
 +
<h4>Transmission electron microscopy</h4>
 +
 
 +
<p>
 +
In order to determine the persistence lenght of the nanowires, we wanted to visualize the nanowires produced by our engineered bacteria. To be able to do so we used TEM to image the cells.
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/d/d3/TEM_7300x_no_induction.png" width="40%" height="100%">
 +
<img src="https://static.igem.org/mediawiki/parts/a/ac/TEM_7300x%2Binduction.png" width="40%" height="100%">
 +
<figcaption>
 +
<b>Fig. 5</b>: TEM images (magnification 7300 x). The left picture shows  uninduced cells  (0% Rhamnose).The picture on the right shows  cells incubated with 1% (w/v) rhamnose.
 +
</figcaption>
 +
</figure>
 +
 
 +
</p>
 +
 
 +
<p>
 +
We did not observe formation of curli nanowires in the uninduced cultures of our strain. However, cells from induced cells clearly produced them, as supported by the TEM images.
 +
</p>
 +
 
 +
</html>
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->
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<partinfo>BBa_K1583000 parameters</partinfo>
 
<partinfo>BBa_K1583000 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
<h3>Reference</h3>
 +
<p>Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, BMC Biotechnology 2008, 8:2 </p>
 +
<p>Zhou, Kang, Kangjian Qiao, Steven Edgar, and Gregory Stephanopoulos. 2015. “Distributing a Metabolic Pathway among a Microbial Consortium Enhances Production of Natural Products.” Nature Biotechnology 33(4): 377–83. </p>

Revision as of 08:55, 18 September 2015

CsgA

This part is meant to express the csgA gene under control of L-rhamnose-inducible promoter. CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. This protein is transported as an unfolded protein out of the cell. Outside the cell CsgA proteins self-assemble into nanowires after nucleation on the membrane protein CsgB. CsgC prevents CsgA proteins from self-assembling inside the cell and the transport is ensured by the proteins CsgEFG. This part was designed to be able to control the nanowire formation.

Characterization

This part was characterized in three different experiments:

  • Fluorescence assay
  • Crystal Violet assay
  • Transmission electron microscopy

    • Fluorescence assay

    To be able to ensure that CsgA is expressed, we added a gene encoding for GFPmut3 (BBa_I13504) under induction of the same rhamnose promoter (BBa_K1583112) to check that the promoter works. In this experiment, the fluorescence signal of our csgA construct and csgA-GFP (I13504) constructs was recorded in time after induction with no, 0.2% (w/v) or 0.5% (w/v) rhamnose. Besides the fluorescence, the OD600 was measured in order to normalize the fluorescence signal per cell.All conditions were carried out in triplicates to be able to do a statistical analysis on the data. The different experiments were induced in a 96 well plate. The OD600 and fluorescence signal was recorded in a plate reader during a 18 hour period of induction at 30°C.

    In Fig. 1, the fluorescent signal was normalized by the number of cells and plotted as a function of time. The red bars denote the error within each ID.

    Fig. 1: Fluorescence signal normalized by the number of cells for 0% (w/v), 0.2% (w/v) and 0.5% (w/v) rhamnose with the csgA and csgA-GFPmut3 construct. The error bars are included for all experiments.

    As can be seen from Fig. 1, only the experiments with 0.2% (w/v) and 0.5% (w/v) rhamnose induction with the csgA-GFPmut3 construct gave a clear increase in fluorescence signal in time. All other experiments, gave similar levels of fluorescence, slightly increasing in time. Furthermore, it can be seen that a higher induction level of rhamnose leads to an increase in GFPmut3 and thus fluorescence. Finally, as the fluorescence signal is normalized by the cell density, one can make statements about the activity of the rhamnose promoter. The promoter seems to not be active right after induction, but more after 3 or 4 hours. This is in accordance with data from literature (Wegerer et. al), in which a low amount of fluorescence with a rhamnose promoter was observed after 2 hours of induction.

    With this kinetic experiment, we have proven that the rhamnose promoter does indeed induce the expression of the csgA gene.

    Crystal violet assay

    The assay above showed that the bacteria that we engineered for the project is capable of producing the CsgA proteins after induction with L-rhamnose. However, this did not yet prove that curli are formed. In order to assess whether our CsgA-producing bacteria can produce these nanowires, our team adapted the protocol from Zhou et al. (2013) that employs crystal violet (methyl violet 10B) for dying the biofilm-making bacteria that attaches to the surface. In the experiment, our CsgA-producing strain of E. coli is induced at a high (0.5% w/v), low (0.2% w/v) and no (0% w/v) concentration of L-rhamnose. Furthermore, csgA deficient bacteria transformed with an empty plasmid (pSB1C3) are used as control.

    Fig. 3: 96-well plate used to test the curli formation capability of our designed constructs. The first two rows (A-B) contained cultures with 0% w/v of rhamnose. The columns D-E and G-H were inducted with L-rhamnose to a final concentration of 0.2% (w/v) and 0.5% (w/v) respectively. The green boxes show the strains carrying BBa_K1583100, and the red ones the control strain with pSB1C3.

    In the end, the wells were diluted with ethanol so all the content can dissolve in the liquid phase. We measured the absorbance at 590 nm of wavelength for all the samples, obtaining the following results (figure 4.).

    Fig. 4: Microtiter Plate Assay results for testing curli formation. CTRL0, CTRL2 and CTRL5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells with pSB1C3, used as a control. CSGA0, CSGA2 and CSGA5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells with BBa_K1583000. The termination 0, 2 and 5 denote the induction with no rhamnose (0), 0.2% w/v (2) and 0.5% w/v (5).

    The cells induced with l-rhamnose showed an increased absorbance at 590 (the peak of the dye) when compared to the empty-plasmid controls, and also with the non-induced sample. That results clearly demonstrate how our engineered cells with BBa_K1583100 successfully make biofilms, regarding that the empty plasmid controls and the analysed samples have significantly (Table 1.) higher crystal violet retention. On the other hand, a higher concentration of rhamnose is not leading to a higher expression. The cells induced with a 0.2% w/v of rhamnose seem to create better a biofilm structure. However, this could be a consequence of different growth patterns; the cultures induced with 0.5% w/v of rhamnose could have stopped duplicating earlier, so the cell concentration could have decreased. So, the dyed area could be consequently smaller.

    Sample p-value Significant difference (10%)
    CTRL2 & CSGA2 0.0426 Yes
    CTRL5 & CSGA5 0.0646 Yes
    CTRL0 & CTRL2 0.5034 No
    CTRL0 & CTRL5 0.9113 No


    With the experiments described previously we achieved nanowire formation with strains containing the BBa_K1583100 biobrick.

    Transmission electron microscopy

    In order to determine the persistence lenght of the nanowires, we wanted to visualize the nanowires produced by our engineered bacteria. To be able to do so we used TEM to image the cells.

    Fig. 5: TEM images (magnification 7300 x). The left picture shows uninduced cells (0% Rhamnose).The picture on the right shows cells incubated with 1% (w/v) rhamnose.

    We did not observe formation of curli nanowires in the uninduced cultures of our strain. However, cells from induced cells clearly produced them, as supported by the TEM images.


    Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      COMPATIBLE WITH RFC[21]
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      COMPATIBLE WITH RFC[1000]


    Reference

    Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, Angelika Wegerer, Tianqi Sun and Josef Altenbuchner, BMC Biotechnology 2008, 8:2

    Zhou, Kang, Kangjian Qiao, Steven Edgar, and Gregory Stephanopoulos. 2015. “Distributing a Metabolic Pathway among a Microbial Consortium Enhances Production of Natural Products.” Nature Biotechnology 33(4): 377–83.