Difference between revisions of "Part:BBa K1583112"
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<p>This part was created to test the <b>functionality</b> of the rhamnose promoter and to measure intracellular <b>steady state</b> concentrations at different induction levels of rhamnose.
 
<p>This part was created to test the <b>functionality</b> of the rhamnose promoter and to measure intracellular <b>steady state</b> concentrations at different induction levels of rhamnose.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1583112 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K1583112 parameters</partinfo>
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<h3>Characterization</h3>
 
<h3>Characterization</h3>
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<h4> Fluorescence assay </h4>
 
<h4> Fluorescence assay </h4>
 
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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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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.  
 
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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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As can be seen from Fig. 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.
 
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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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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.
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<h4> Crystal violet assay </h4>
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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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<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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<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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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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<img src="https://static.igem.org/mediawiki/parts/1/1b/Crystal_Violet_result_K1583100.JPG" width="70%" height="100%">
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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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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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|'''CTRL2 & CSGA2'''
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|Yes
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|'''CTRL5 & CSGA5'''
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|'''CTRL0 & CTRL2'''
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|'''CTRL0 & CTRL5'''
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With the experiments described previously we achieved nanowire formation with strains containing the BBa_K1583100 biobrick.
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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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Using TEM the formation of curli of the biobrick <a href="https://parts.igem.org/Part:BBa_K1583100">BBa_K1583100</a> was visualized. 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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<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%">
 
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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.  
 
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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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<!-- -->
 

Revision as of 18:47, 18 September 2015

pRha + CsgA & GFP in same operon

CsgA is a protein monomer which can aggregate to form amyloid nanowires in natural biofilms of E.coli. The aggregation to the nanowire ('curli') is induced by the membrane protein CsgB. CsgC, CsgE, CsgF and CsgG act as chaperones for CsgA during translation and export CsgA to the extracellular space.

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 which is under control of the Rhamnose promoter.

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.
On the way there, many questions need to be answered. To do this in the most efficient way, we went back to good old modeling.

Transcriptional and translational rates can be approximated with reasonable precision. However, we wanted to measure this!

To do so, we created this device. The beginning of the device consists of our standard parts: the rhamnose promoter and the CsgA gene.
However, we skipped the terminator behind the gene.

In this manner we were able to clone the gene from the biobrick BBa_I13504 coding for GFP behind CsgA and into the same operon.
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 fluorescence [au] and mass [ng] we were able to calculate the expression rate of CsgA with units [proteins/(cell*second)].

This part was created to test the functionality of the rhamnose promoter and to measure intracellular steady state concentrations at different induction levels of rhamnose. 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 three different experiments:

  • Fluorescence assay
  • Crystal Violet 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 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 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. 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.