Difference between revisions of "Part:BBa K1583112"
 
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<p><i>CsgA</i> is a protein monomer which can aggregate to form amyloid nanowires in natural <i>biofilms</i> of E.coli.
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This part is meant to express the <i>csgA</i> and <i>GFPmut3</i> gene under control of L-rhamnose-inducible promoter.
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The aggregation to the nanowire ('curli') is induced by the membrane protein <i>CsgB. CsgC, CsgE, CsgF</i> and <i>CsgG</i> act as chaperones for CsgA during translation and export CsgA to the extracellular space.</p>
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<p>This part was designed to measure intracellular expression rates of CsgA coupled to fluorescence (GFP) by cloning the biobrick <a href="https://parts.igem.org/Part:BBa_I13504">BBa_I13504</a> into the same operon which is under control of the <i>Rhamnose promoter</i>.</p>
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<p>A biofilm is created, when sufficient levels of mature CsgA are present in the extracellular space. CsgB can then act as a nucleator inducing the curli formation.<br> On the way there, many questions need to be answered.
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<p>CsgA is a protein monomer which can aggregate to form amyloid fibers in natural biofilms of <i>E.coli</i>. 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 its transport is ensured by the proteins Csg-E-F-G.</p>
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To do this in the most efficient way, we went back to good old <b>modeling</b>.</p>
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<p>Transcriptional and translational rates can be approximated with reasonable precision. However, we wanted to measure this!</p>
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<p>This part was designed to measure intracellular expression rates of CsgA coupled to fluorescence (GFP) by cloning the biobrick <a href="https://parts.igem.org/Part:BBa_I13504"target=_blank">BBa_I13504</a> into the same operon in up-stream of a Rhamnose promoter.</p>
  
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<p>To do so, we created this device. The beginning of the device consists of our standard parts: the rhamnose promoter and the CsgA gene. <br> However, we skipped the terminator behind the gene. </p>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
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<partinfo>BBa_K1583112 SequenceAndFeatures</partinfo>
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<p>In this manner we were able to clone the gene from the biobrick BBa_I13504 coding for GFP behind CsgA and <b>into the same operon</b>.<br>
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<!-- Uncomment this to enable Functional Parameter display
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We assume that the difference between CsgA and CsgA+GFP in size does not influence transcription and translation of CsgA. Using a calibration curve connecting <b>fluorescence</b> [au] and <b>mass</b> [ng] we were able to calculate the expression rate of CsgA with units [proteins/(cell*second)].</p>
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===Functional Parameters===
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<partinfo>BBa_K1583112 parameters</partinfo>
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<h3>Characterization</h3>
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<p><h3>Characterization</h3></p>
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<p>
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<p>This part was characterized in two different experiments:
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This part was characterized in three different experiments:
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<ul><li> Fluorescence assay </li>
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<li> Fluorescence assay </li>
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<li> Transmission electron microscopy </li></ul></p>
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<li> Crystal Violet assay </li>
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<li> Transmission electron microscopy </li>
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</p>
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<h4> Fluorescence assay </h4>
 
<h4> Fluorescence assay </h4>
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<p>
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<p>To be able to ensure that CsgA is expressed, we used this biobrick to check that the rhamnose inducible promoter works. In this experiment, the fluorescence signal of our CsgA construct and CsgA-GFP construct 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>
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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">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.  
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</p>
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<p>
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<p>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. </p>
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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.  
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</p>
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<figure>
 
<figure>
 
<img src="https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png" width="100%" height="100%">
 
<img src="https://static.igem.org/mediawiki/2015/d/d3/Modelling_pic_9_.png" width="100%" height="100%">
 
<figcaption>
 
<figcaption>
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<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.  
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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.  
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
  
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<p>
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<p>As can be seen from figure 1, only the experiments with 0.2% (w/v) and 0.5% (w/v) rhamnose induction with this biobrick 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>
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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.
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</p>
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<p>
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<p>With this kinetic experiment, we have proven that the rhamnose promoter does indeed induce the expression of the <i>csgA</i> gene and the I13504 gene.</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.
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</p>
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<h4> Crystal violet assay </h4>
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<p>
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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.
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</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/parts/4/46/96_wells_plate_Crystal_Violet_K1583100.jpg" width="50%" height="100%">
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<figcaption>
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<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.
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</figcaption>
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</figure>
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<p>
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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.).
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</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/parts/1/1b/Crystal_Violet_result_K1583100.JPG" width="70%" height="100%">
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<figcaption>
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<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).
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</figcaption>
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</figure>
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<p>
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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.
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{|style="color:black" cellpadding="4" 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 (10%)'''</FONT>
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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
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|Yes
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|'''CTRL0 & CTRL2'''
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|0.5034
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|No
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|-
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|'''CTRL0 & CTRL5'''
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|0.9113
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|No
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|}
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<html>
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<!--Table formatting from https://parts.igem.org/Part:BBa_K1150020 -->
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<p>
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With the experiments described previously we achieved nanowire formation with strains containing the BBa_K1583100 biobrick.
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</p>
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<h4>Transmission electron microscopy</h4>
 
<h4>Transmission electron microscopy</h4>
  
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<p>
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<p>Using TEM the formation of curli of the biobrick <a href="https://parts.igem.org/Part:BBa_K1583100">BBa_K1583100</a> was visualized (Figure 2). Although this is a different biobrick (no GFP gene behind the same promoter), curli formation can be presumed to be similar for this biobrick.</p>
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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.
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<figure>
 
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<img src="https://static.igem.org/mediawiki/parts/a/ac/TEM_7300x%2Binduction.png" width="40%" height="100%">
 
<img src="https://static.igem.org/mediawiki/parts/a/ac/TEM_7300x%2Binduction.png" width="40%" height="100%">
 
<figcaption>
 
<figcaption>
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<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.  
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<b>Figure 2</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>
 
</figcaption>
 
</figure>
 
</figure>
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</p>
 
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<p><h3>References</h3></p>
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<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>
 
</html>
 
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<!-- -->
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K1583112 SequenceAndFeatures</partinfo>
 
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<!-- Uncomment this to enable Functional Parameter display
 
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===Functional Parameters===
 
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<partinfo>BBa_K1583112 parameters</partinfo>
 
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<!-- -->
 

Latest revision as of 15:30, 13 November 2015

pRha + CsgA & GFP in same operon

This part is meant to express the csgA and GFPmut3 gene under control of L-rhamnose-inducible promoter.

CsgA is a protein monomer which can aggregate to form amyloid fibers 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 its transport is ensured by the proteins Csg-E-F-G.

This part was designed to measure intracellular expression rates of CsgA coupled to fluorescence (GFP) by cloning the biobrick BBa_I13504 into the same operon in up-stream of a Rhamnose promoter.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1308


Characterization

This part was characterized in two different experiments:

  • Fluorescence assay
  • Transmission electron microscopy

Fluorescence assay

To be able to ensure that CsgA is expressed, we used this biobrick to check that the rhamnose inducible promoter works. In this experiment, the fluorescence signal of our CsgA construct and CsgA-GFP construct 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 this biobrick 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 and the I13504 gene.

Transmission electron microscopy

Using TEM the formation of curli of the biobrick BBa_K1583100 was visualized (Figure 2). Although this is a different biobrick (no GFP gene behind the same promoter), curli formation can be presumed to be similar for this biobrick.

Figure 2: 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.

References

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