Difference between revisions of "Part:BBa K1583111"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1583111 short</partinfo>
 
<partinfo>BBa_K1583111 short</partinfo>
  
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The fusion protein of CsgA with Mfp3 and N-terminal His tag was improved by insertion of constitutively expressed RFP in a different operon to make the cells red fluorescent.
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<html>
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<p>This part is meant to express a fusion protein of the <i>csgA</i> and the <i>Mfp3</i> gene with His-tag under control of L-rhamnose-inducible promoter. Addionally a RFP coding device was added (<a href="https://parts.igem.org/Part:BBa_I13521"target="_blank">BBa_I13521</a>).
  
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<p>CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from <i>E.coli K-12 MG1655</i>. Inspired by mussels, the Mfp3 (mussel foot protein) has high adhesive properties towards wet polar surfaces. 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. By creating a fusion protein of CsgA and Mfp3, the adhesive properties of the mussel foot protein is combined with the formation of nanowires.
  
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<!-- Add more about the biology of this part here
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<p>The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres" (Zhong et al, 2014).</p>
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===Usage and Biology===
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</html>
 
<!-- -->
 
<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
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<partinfo>BBa_K1583111 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1583105 SequenceAndFeatures</partinfo>
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<html>
  
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<h3>Characterization</h3>
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<p>
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Three different experiments were done to characterise the BBa_K1583105 biobrick:
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<ul><li> Fluorescence assay </li>
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<li> Crystal Violet assay </li>
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<li> Transmission electron microscopy </li></ul>
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As shown in the crystal violet assay in the characterization section of pRha+CsgA & pTet+RFP (<a href="https://parts.igem.org/Part:BBa_K1583106"target="_blank">BBa_K1583106</a>), the RFP conding device does not affect the formation of CsgA protein. Therefore the characterization data of <a href="https://parts.igem.org/Part:BBa_K1583105"target="_blank">BBa_K1583105</a> is also valid for this biobrick.</p>
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<h4> Fluorescence assay </h4>
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<p>
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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"target="_blank">BBa_I13504</a>) under induction of the same rhamnose promoter (<a href="https://parts.igem.org/Part:BBa_K1583112"target="_blank">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.
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</p>
  
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<!-- Uncomment this to enable Functional Parameter display  
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<p>
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===Functional Parameters===
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In figure 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.
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<partinfo>BBa_K1583111 parameters</partinfo>
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</p>
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<!-- -->
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<figure>
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<img src="https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png" width="100%" height="100%">
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<figcaption>
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<b>Figure 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.
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</figcaption>
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</figure>
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<p>
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As can be seen from figure 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>
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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 bacteria producting the fusion protein CsgA_Mfp3_His can still 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_Mfp3_His-producing strain of <i>E. coli</i> was 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) were used as control. 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 2.).
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</p>
 +
 
 +
<figure><img class="featurette-image img-responsive center-block" src="https://static.igem.org/mediawiki/2015/a/a9/TU_Delft_mfp3.png
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" style="width:70%; background-size: cover;" alt="Generic placeholder image"><figcaption><b>Figure 2</b>: Microtiter Plate Assay results for testing biofilm formation. All the measurements were conducted in triplicates.  CTRL0, CTRL2 and CTRL5 are <i>E. coli K-12 MG1655 PRO ΔcsgA ompR234</i> cells with pSB1C3, used as a control. MFP30, MFP32 and MFP35 are <i>E. coli K-12 MG1655 PRO ΔcsgA ompR234</i> cells expressing a plasmid that contains csgA attached to the mfp3 peptide under an inducible promoter. 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>
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<p> To confirm that there is a real change between the analysed samples and the empty plasmid control, a significance analysis was performed for α=0.05 (Table 1.).</p>
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<br><ul><caption>
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<b>Table 1</b>: Significance analysis of the samples analysed, with a significance α value of 5%. All the samples display a significant difference when compared to the empty plasmid homologe sample (CTRL). 
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</caption>
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</html>
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{|style="color:black" cellpadding="6" cellspacing="2" border="1" align="middle"
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! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''Sample'''</FONT>
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! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''p-value'''</FONT>
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! style="background:#0084A7;"|<FONT COLOR="#FFFFFF">'''Significant difference (5%)'''</FONT>
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|-
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|'''CTRL2 & MFP32'''
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|0.0127
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|Yes
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|-
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|-
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|'''CTRL5 & MFP35'''
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|0.0170
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|Yes
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|}
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<html></ul><br> <!--Table formatting originally from https://parts.igem.org/Part:BBa_K1150020 -->
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<p>
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The significance analysis shows that cells containing the CsgA_Mfp3_His (BBa_K1583105) biobrick can efficiently create a curli, when compared with an empty plasmid control (i.e. without csgA expression).
 +
</p> <br>
 +
 
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<h4>Transmission electron microscopy</h4>
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 +
<p>
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Using TEM the formation of curli of the biobrick <a href="https://parts.igem.org/Part:BBa_K1583100">BBa_K1583100</a> was visualized (Figure 3). Although this is a different biobrick containing only CsgA (compared to the fusion protein of Mfp3_CsgA_His), curli formation can be presumed to be similar for this biobrick suggested by the Crystal Violet assay.</p>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/d/d3/TEM_7300x_no_induction.png" width="40%" height="100%">
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<img src="https://static.igem.org/mediawiki/parts/a/ac/TEM_7300x%2Binduction.png" width="40%" height="100%">
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<figcaption>
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<b>Figure 3.</b>: TEM images (magnification 7300 x) of cells containing <a href="https://parts.igem.org/Part:BBa_K1583100"target="_blank">BBa_K1583100</a>. The left picture shows  uninduced cells (0% Rhamnose).The picture on the right shows  cells incubated with 1% (w/v) rhamnose.
 +
</figcaption>
 +
</figure>
 +
 
 +
<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>
 +
 
 +
<h3>Reference</h3>
 +
<p>Wegerer, A., Sun, T., and Altenbuchner, J. (2008). Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, BMC Biotechnology, 8:2 </p>
 +
Zhong, C. et al., 2014. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature nanotechnology, 9(10), pp.858–66</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>
 +
</html>

Latest revision as of 22:50, 18 September 2015

pRha + CsgA_Mfp3_His fusion protein & pTET + RFP

This part is meant to express a fusion protein of the csgA and the Mfp3 gene with His-tag under control of L-rhamnose-inducible promoter. Addionally a RFP coding device was added (BBa_I13521).

CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms taken from E.coli K-12 MG1655. Inspired by mussels, the Mfp3 (mussel foot protein) has high adhesive properties towards wet polar surfaces. 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. By creating a fusion protein of CsgA and Mfp3, the adhesive properties of the mussel foot protein is combined with the formation of nanowires.

The design was based on the paper "Strong underwater adhesives made by self-assembling multi-protein nanofibres" (Zhong et al, 2014).

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]

Characterization

Three different experiments were done to characterise the BBa_K1583105 biobrick:

  • Fluorescence assay
  • Crystal Violet assay
  • Transmission electron microscopy
As shown in the crystal violet assay in the characterization section of pRha+CsgA & pTet+RFP (BBa_K1583106), the RFP conding device does not affect the formation of CsgA protein. Therefore the characterization data of BBa_K1583105 is also valid for this biobrick.

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 figure 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.

Figure 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 figure 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 bacteria producting the fusion protein CsgA_Mfp3_His can still 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_Mfp3_His-producing strain of E. coli was 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) were used as control. 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 2.).

Generic placeholder image
Figure 2: Microtiter Plate Assay results for testing biofilm formation. All the measurements were conducted in triplicates. CTRL0, CTRL2 and CTRL5 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells with pSB1C3, used as a control. MFP30, MFP32 and MFP35 are E. coli K-12 MG1655 PRO ΔcsgA ompR234 cells expressing a plasmid that contains csgA attached to the mfp3 peptide under an inducible promoter. The termination 0, 2 and 5 denote the induction with no rhamnose (0), 0.2% w/v (2) and 0.5% w/v (5).

To confirm that there is a real change between the analysed samples and the empty plasmid control, a significance analysis was performed for α=0.05 (Table 1.).


    Table 1: Significance analysis of the samples analysed, with a significance α value of 5%. All the samples display a significant difference when compared to the empty plasmid homologe sample (CTRL).

    Sample p-value Significant difference (5%)
    CTRL2 & MFP32 0.0127 Yes
    CTRL5 & MFP35 0.0170 Yes


The significance analysis shows that cells containing the CsgA_Mfp3_His (BBa_K1583105) biobrick can efficiently create a curli, when compared with an empty plasmid control (i.e. without csgA expression).


Transmission electron microscopy

Using TEM the formation of curli of the biobrick BBa_K1583100 was visualized (Figure 3). Although this is a different biobrick containing only CsgA (compared to the fusion protein of Mfp3_CsgA_His), curli formation can be presumed to be similar for this biobrick suggested by the Crystal Violet assay.

Figure 3.: TEM images (magnification 7300 x) of cells containing BBa_K1583100. 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.

Reference

Wegerer, A., Sun, T., and Altenbuchner, J. (2008). Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations”, BMC Biotechnology, 8:2

Zhong, C. et al., 2014. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature nanotechnology, 9(10), pp.858–66

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.