Difference between revisions of "Part:BBa K4757062"
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<partinfo>BBa_K4757062 short</partinfo>
 
<partinfo>BBa_K4757062 short</partinfo>
  
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Terephthalic acid sensing transcription factor - fluorescence mKate2 expression cassette.....
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<html>
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to be continued...
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<head>
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    <titl>BBa_K4757062</titl>
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</head>
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<body>
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    <p>Contents</p>
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    <p><a href="#Usage_and_Biology">1. Usage and Biology</a></p>
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    <p><a href="#section2">2. Results</a></p>
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    <h1 id="#Usage_and_Biology"><a>1. Usage and Biology</a></h1>
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    <p>The PET degradation product terephthalic acid (TPA) is monitored by the XylS-K38R-L224Q (<i>XylS-mt</i>)
 +
        transcription factor. Li <i>et al.</i> discovered two point mutations K38R and L224Q makes XylS sensitive to TPA
 +
        in concentrations as low as 10 µM in <i>E. coli</i> (Li <i>et al.</i>, (2022)). Upon activation with TPA or the
 +
        well described XylS inducer 3-methyl-benzoate (MBA), XylS-mt dimerizes and binds the <i>Pm</i> promoter (Gawin
 +
        <i>et al.</i>, 2017). <i>Pm</i> activation results in the expression of small regulatory RNAs (sRNAs), capable
 +
        of blocking the translation of the GOI. A negative feedback loop is established, downregulating the GOI activity
 +
        at high PET depolymerization rates.</p>
 +
    <p>The expression of XylS-mt itself is regulated through the <i>Ps1/Ps2</i> promoter (Gallegos <i>et al.</i>, 1996;
 +
        Gawin <i>et al.</i>, 2017). In the absence of TPA, a low baseline of XylS-mt is present in the cell through
 +
        constitutive low expression from the <i>Ps2</i> promoter. However, upon XylS-mt activation the transcription
 +
        factor also binds the <i>Ps1</i> promoter leading to high levels of induction (Gallegos <i>et al.</i>, 1996).
 +
        This is the first time a TPA sensor is characterized in <i>P. fluorescens</i> and in the iGEM parts registry.
 +
    </p>
 +
    <p>&nbsp;</p>
 +
    <h1 id="#section2"><a>2. Results</a></h1>
 +
    <h3 id="XylS-WT_TPA_sensitivity_testing"><a>2.1 XylS-WT TPA sensitivity testing</a></h3>
 +
    <p>The XylS-mt sensitivity towards TPA was compared to the XylS-WT sensitivity. XylS-WT showed no sensitivity
 +
        towards TPA and good sensitivity towards MBA. When comparing the sensitivities of XylS-mt and XylS-WT to MBA,
 +
        the introduced mutations seemed to cause a 60-70 % decrease in expression strength (figure 4).</p>
 +
    <figure class="figure">
 +
        <img src="https://static.igem.wiki/teams/4757/wiki/induction-strength-comparison-xylswt-vs-xylsmt.svg"
 +
            alt="image004">
 +
        <figcaption class="figcaption img-caption"> <strong>Figure 4: Comparison of expression strength of wildtype and
 +
                mutated (K38R, L224Q) XylS, at three different inducer concentrations.</strong>
 +
            <br>Values are presented as mean +/-SD. No statistical analysis was performed.
 +
        </figcaption>
 +
    </figure>
 +
    <p>&nbsp;</p>
 +
    <h2 id="XylS-mt_induction_with_XylR_activation"><a>4.2 XylS-mt induction with XylR activation</a></h2>
 +
    <p>Co-induction with varying concentrations of TPA and m-Xylene or TPA and Toluene (5 nM, 50 nM, 500 nM m-Xylene or
 +
        Toluene mixed with 0 nM, 2.5 nM, 5 nM, 10 nM, 50 nM, 500 nM, or 1 mM TPA) was tested to improve the induction of
 +
        XylS-mt and the expression of the GOI. Toluene and Xylene are inductors of the genomic transcription factor
 +
        XylR, previously described to jointly activate expression from the <i>Ps1</i> promoter with XylS in <i> P.
 +
            putida</i>. However, co-induction showed no increase in expression strength (data not shown).</p>
 +
    <p>&nbsp;</p>
 +
    <h2 id="5.2.2."><a>5.3 Ps1/Ps2 XylS-MT TPA and MBA co-induction</a></h2>
 +
    <p>To further test the influence of the Ps1/Ps2 promoter system on XylS-mt, the co-induction was tested with
 +
        previously determined MBA and TPA concentrations. Three TPA concentrations were tested with one of four MBA
 +
        concentrations. Fold change and normalized fluorescence were calculated (Figure 13). At an MBA concentration of
 +
        0.0025 mM, a significant TPA dependent fold change could be measured (1.29 +/- 0.056, p < 0.001). Higher MBA
 +
            concentrations (0.0075 mM MBA, 0.015 mM MBA) showed an overall decreased fold change. Decrease after TPA
 +
            induction is due to referencing errors caused by TPA precipitation. The expression strength shows an overall
 +
            decreased fluorescence intensity at low MBA concentrations, despite co-induction with TPA (Figure 13,
 +
            right)</p>
 +
            <figure class="figure">
 +
                <img style="width: 500% !important;
 +
display: block;
 +
margin-left: auto;
 +
margin-right: auto;"
 +
                    src="https://static.igem.wiki/teams/4757/wiki/xyls-tpa-sensitivity-of-ps1ps2-xylsmt-upon-mba-activation-expression-strength-of-ps1ps2-upon-induction-with-tpa-and-mba.svg"
 +
                    alt="image021">
 +
                <figcaption class="figcaption img-caption">
 +
                    <strong>Figure 13: Expression strength with TPA and MBA co-induction</strong>
 +
                    <br>Left: Fold change in expression of different MBA inducer concentrations after co-induction of
 +
                    TPA
 +
                    <br>Right: Expression strength measured in relative fluorescence of different MBA inducer
 +
                    concentrations after co-induction of varying TPA concentrations.
 +
                    <br>Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 mg/mL MBA
 +
                    or 0 mg/mL TPA at each time point. Reported significances were determined with ordinary One-way
 +
                    ANOVA with Dunn's method for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p <
 +
                        0.0001. To reduce complexity only significant test results are shown. </figcaption>
 +
            </figure>
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            <p>&nbsp;</p>
 +
    <h1 id="Engineering"><a>3. Engineering</a></h1>
 +
    <h2 id="XylS_-_cloning"><a>3.1 XylS - cloning</a></h2>
 +
    <p>Since the XylS/Pm expression system is natively found on the pSEVA438 plasmid only the two point mutations, K38R
 +
        and L224Q, needed to be introduced. Two primer pairs were used to add the single base pair substitutions. The
 +
        sensitivity of XylS-mt towards was studied using the native Ps1/Ps2 promoter system but found to yield low
 +
        expression levels in the TPA sensitive range (<a href="#5.2.">see section 5.2</a>). To mitigate this problem,
 +
        the Ps1/Ps2 promoter system was substituted with pEM7 to further test the functionality in different scenarios.
 +
        The fluorescence reporter gene mKate2 was cloned with SacI and PstI into the MCS downstream of Pm, add-on PCR
 +
        was used to introduce the Anderson library promoter RBS BBa_J61100. (Figure 3).</p>
 +
    <figure class="figure">
 +
        <img style="width: 80% !important;
 +
        display: block;
 +
        margin-left: auto;
 +
        margin-right: auto;" src="https://static.igem.wiki/teams/4757/wiki/xyls-overview-mew.svg" alt="XylS_mew">
 +
        <p>&nbsp;</p>
 +
        <figcaption class="figcaption img-caption"><i><strong>Figure 3: Overview of genetic construct for XylS-MT
 +
                    testing</strong></i> </figcaption>
 +
    </figure>
 +
    <p>&nbsp;</p>
 +
   
 +
 +
</body>
 +
 +
</html>
 
Engnineering journey
 
Engnineering journey
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<p>Mutation -> weak expression/no expression after mutation -> enhancing with RBS -> still weak/no expression -> co-induction -> significant changes could be detected </p>
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<p>Mutation -> weak expression/no expression after mutation -> enhancing with RBS -> still weak/no expression ->
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<p> co-induction showed so low exprssion -> pEM7 promoter was used to substitute Ps1/Ps2 -> high expression but leaky </p>
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    co-induction -> significant changes could be detected </p>
 +
<p> co-induction showed so low exprssion -> pEM7 promoter was used to substitute Ps1/Ps2 -> high expression but leaky
 +
</p>
 +
 
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4757062 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4757062 SequenceAndFeatures</partinfo>
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Revision as of 09:41, 12 October 2023

_ TPA sensing XylS-K38R-L224Q/Pm expressing mKate2

BBa_K4757062

Contents

1. Usage and Biology

2. Results

1. Usage and Biology

The PET degradation product terephthalic acid (TPA) is monitored by the XylS-K38R-L224Q (XylS-mt) transcription factor. Li et al. discovered two point mutations K38R and L224Q makes XylS sensitive to TPA in concentrations as low as 10 µM in E. coli (Li et al., (2022)). Upon activation with TPA or the well described XylS inducer 3-methyl-benzoate (MBA), XylS-mt dimerizes and binds the Pm promoter (Gawin et al., 2017). Pm activation results in the expression of small regulatory RNAs (sRNAs), capable of blocking the translation of the GOI. A negative feedback loop is established, downregulating the GOI activity at high PET depolymerization rates.

The expression of XylS-mt itself is regulated through the Ps1/Ps2 promoter (Gallegos et al., 1996; Gawin et al., 2017). In the absence of TPA, a low baseline of XylS-mt is present in the cell through constitutive low expression from the Ps2 promoter. However, upon XylS-mt activation the transcription factor also binds the Ps1 promoter leading to high levels of induction (Gallegos et al., 1996). This is the first time a TPA sensor is characterized in P. fluorescens and in the iGEM parts registry.

 

2. Results

2.1 XylS-WT TPA sensitivity testing

The XylS-mt sensitivity towards TPA was compared to the XylS-WT sensitivity. XylS-WT showed no sensitivity towards TPA and good sensitivity towards MBA. When comparing the sensitivities of XylS-mt and XylS-WT to MBA, the introduced mutations seemed to cause a 60-70 % decrease in expression strength (figure 4).

image004
Figure 4: Comparison of expression strength of wildtype and mutated (K38R, L224Q) XylS, at three different inducer concentrations.
Values are presented as mean +/-SD. No statistical analysis was performed.

 

4.2 XylS-mt induction with XylR activation

Co-induction with varying concentrations of TPA and m-Xylene or TPA and Toluene (5 nM, 50 nM, 500 nM m-Xylene or Toluene mixed with 0 nM, 2.5 nM, 5 nM, 10 nM, 50 nM, 500 nM, or 1 mM TPA) was tested to improve the induction of XylS-mt and the expression of the GOI. Toluene and Xylene are inductors of the genomic transcription factor XylR, previously described to jointly activate expression from the Ps1 promoter with XylS in P. putida. However, co-induction showed no increase in expression strength (data not shown).

 

5.3 Ps1/Ps2 XylS-MT TPA and MBA co-induction

To further test the influence of the Ps1/Ps2 promoter system on XylS-mt, the co-induction was tested with previously determined MBA and TPA concentrations. Three TPA concentrations were tested with one of four MBA concentrations. Fold change and normalized fluorescence were calculated (Figure 13). At an MBA concentration of 0.0025 mM, a significant TPA dependent fold change could be measured (1.29 +/- 0.056, p < 0.001). Higher MBA concentrations (0.0075 mM MBA, 0.015 mM MBA) showed an overall decreased fold change. Decrease after TPA induction is due to referencing errors caused by TPA precipitation. The expression strength shows an overall decreased fluorescence intensity at low MBA concentrations, despite co-induction with TPA (Figure 13, right)

image021
Figure 13: Expression strength with TPA and MBA co-induction
Left: Fold change in expression of different MBA inducer concentrations after co-induction of TPA
Right: Expression strength measured in relative fluorescence of different MBA inducer concentrations after co-induction of varying TPA concentrations.
Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 mg/mL MBA or 0 mg/mL TPA at each time point. Reported significances were determined with ordinary One-way ANOVA with Dunn's method for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. To reduce complexity only significant test results are shown.

 

3. Engineering

3.1 XylS - cloning

Since the XylS/Pm expression system is natively found on the pSEVA438 plasmid only the two point mutations, K38R and L224Q, needed to be introduced. Two primer pairs were used to add the single base pair substitutions. The sensitivity of XylS-mt towards was studied using the native Ps1/Ps2 promoter system but found to yield low expression levels in the TPA sensitive range (see section 5.2). To mitigate this problem, the Ps1/Ps2 promoter system was substituted with pEM7 to further test the functionality in different scenarios. The fluorescence reporter gene mKate2 was cloned with SacI and PstI into the MCS downstream of Pm, add-on PCR was used to introduce the Anderson library promoter RBS BBa_J61100. (Figure 3).

XylS_mew

 

Figure 3: Overview of genetic construct for XylS-MT testing

 

Engnineering journey

Mutation -> weak expression/no expression after mutation -> enhancing with RBS -> still weak/no expression -> co-induction -> significant changes could be detected

co-induction showed so low exprssion -> pEM7 promoter was used to substitute Ps1/Ps2 -> high expression but leaky


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 212
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 934
  • 1000
    COMPATIBLE WITH RFC[1000]