Difference between revisions of "Part:BBa K3187015"
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                     target="_blank"> BBa_B0032</a>). The improved and the original version of the part were cloned into the pSB1C3 backbone and transformed in <i>E. coli</i> BL21 (DE3).  
 
                     target="_blank"> BBa_B0032</a>). The improved and the original version of the part were cloned into the pSB1C3 backbone and transformed in <i>E. coli</i> BL21 (DE3).  
 
             </p>
 
             </p>
−
       
+
Both parts were improved in terms of their expression levels, based on the insertion of a 5’ untranslated region (5’-UTR) upstream of the coding sequence. This 5'-UTR was adapted from iGEM Bielefeld 2015 (&nbsp;<a
−
            <h3>Results</h3>
+
                  href="https://parts.igem.org/Part:BBa_K1758100"
−
            <hr class="head">
+
                    target="_blank"> BBa_K1758100</a>) and is based on the research of Olins <i>et</i> al
 +
<sup id="cite_ref-1" class="reference">
 +
                        <a href="#cite_note-1">[1] </a>
 +
                    </sup>
 +
and Takahashi <i>et</i> al.
 +
<sup id="cite_ref-2" class="reference">
 +
                        <a href="#cite_note-2">[2] </a>
 +
                    </sup>.
 +
It contains the strong ribosomal binding site (RBS) g10-L from the T7 bacteriophage and a sequence that plays a role in the regulation of mRNA binding to and release from the 30S ribosomal subunit. The 5'-UTR therefore enhances the translation efficiency of the following coding sequence (CDS) (Fig. 1).
  
−
            <h4>Cloning</h4>
+
      </p>
  
−
            <p>
+
<img
−
                pTeTW3con2-ptet-mCherry--sfGFP-pT7 was cloned in two steps via a restriction and
+
        class="img-fluid center"
−
                ligation protocol. First, the mCherry gene was cloned into the backbone pTeTW3con2. Sequencing analysis
+
        src="https://2019.igem.org/wiki/images/6/62/T--TU_Darmstadt--RibosomLiberation.png"
−
                was carried out to test whether the cloning was positive before the next step started. Next, the sfGFP
+
        style="max-width:80%"
−
                gene was cloned into the backbone (pTeTw3con2-ptet-mCherry). The cloning fo the final product was
+
      />
−
                checked via sequencing.
+
<div class="caption">
−
            </p>
+
                                                        <p>
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            <br></br>
+
                                                            <b>
−
            <h4>Measuring the expression levels after single induction</h4>
+
                                                                <center> Figure 1:
−
 
+
    </b> Schematic depiction of the composition and interaction of the enhancer sequence with the 30S ribosomal subunit described by Takahashi <i>et.</i> al. <sup id="cite_ref-2" class="reference">
−
 
+
                         <a href="#cite_note-2">[2] </a>
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            <p>
+
                     </sup>.
−
                The measurement (fig. 1 and 2) showed a strong background expression of the T7 site
+
</center>
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                represented
+
                                                        </p>
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                by the increasing fluorescence signal of sfGFP in the uninduced condition.
+
                    </div>
−
                However, this background expression of sfGFP lessend with a rising AHT
+
−
                concentration. This came as quite the surprise, since inducing with
+
−
                different
+
−
                AHT concentrations was supposed to mainly regulate the tetA regulated site.
+
−
                Generally, the data shows a clear excess of the sfGFP fluorescence.
+
−
            </p>
+
−
 
+
−
            <p>
+
−
                Unfortunately, we were not able to select the fitting settings for
+
−
                monitoring
+
−
                the fluorescence signals of sfGFP and mCherry. The final fluorescence signal
+
−
                of
+
−
                sfGFP in IPTG induced triplicates was stronger than the maximal detection
+
−
                point
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−
                of the instrument while a change in the signal of mCherry was barely
+
−
                detected
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−
                during the measuring process.
+
−
            </p>
+
−
 
+
−
            <div class="row">
+
−
 
+
−
                <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
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−
                    <img class="img-fluid center"
+
−
                        src="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Spectramax_AHT_%28Min_Max%29.png"
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                        style="max-width:50%">
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−
                    <div class="caption">
+
−
                        <p>
+
−
                            <b>Figure 1:</b>
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−
                            Spectrophotometric measurement of the fluorescences of mCherry
+
−
                            (red) and sfGFP (blue) triplicates after inducing with AHT. AHT
+
−
                            was induced at 90 minutes. The iduction with 0.1 µg/mL is shown
+
−
                            in light red and blue and 0.3 µg/mL is shown in dark red and
+
−
                            blue.
+
−
                            <a href="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Spectramax_AHT_%28Min_Max%29.png"
+
−
                                target="_blank">View full size image</a>.
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                        </p>
+
−
                    </div>
+
−
                </div>
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−
                <br></br>
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                <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
+
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                    <img class="img-fluid center"
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−
                        src="https://2019.igem.org/wiki/images/7/7c/T--TU_Darmstadt--Spectramax_IPTG_%28Min_Max%29.png"
+
−
                        style="max-width:50%">
+
−
                    <div class="caption">
+
−
                        <p>
+
−
                            <b>Figure 2:</b>
+
−
                            Spectrophotometric measurement of the fluorescences of mCherry
+
−
                            (red)
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−
                            and
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−
                            sfGFP (blue) triplicates after inducing with IPTG. IPTG was
+
−
                            induced
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                            at 90
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−
                            minutes. The
+
−
                            iduction with 0.1 mM is shown in light red and blue and 1 mM is
+
−
                            shown in
+
−
                            dark red and blue. An uninduced sfGFP variant is shown in
+
−
                            orange.
+
−
                            <a href="https://2019.igem.org/wiki/images/7/7c/T--TU_Darmstadt--Spectramax_IPTG_%28Min_Max%29.png"
+
−
                                target="_blank">View full size image</a>.
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−
                        </p>
+
−
                    </div>
+
−
 
+
−
                </div>
+
−
            </div>
+
−
            <br></br>
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−
            <h4>Testing various dual induction strategies</h4>
+
−
 
+
−
 
+
−
            <p>
+
−
                The results of the samples which were collected after 6 h of expression (fig. 3)
+
−
                showed a
+
−
                distinct trend. Whenever IPTG was induced during the experimental procedure
+
−
                there was a large excess of sfGFP in comparison to mCherry. The induction
+
−
                times
+
−
                or used concentrations of IPTG just had a small effect on the resulting
+
−
                ratio of
+
−
                sfGFP to mCherry. Only the variant which was just induced with AHT showed a
+
−
                significant difference having a 2:1 ratio of sfGFP to mCherry. As expected
+
−
                from
+
−
                the previous experiments, the uninduced control showed the highest excess of
+
−
                sfGFP in this series.
+
−
            </p>
+
−
            <br></br>
+
−
            <img class="img-fluid center"
+
−
                src="https://2019.igem.org/wiki/images/d/d5/T--TU_Darmstadt--Ratio_of_sfGFPmCherry_after_6_h.png"
+
−
                style="max-width:50%" />
+
−
 
+
−
            <div class="caption">
+
−
                <p>
+
−
                    <b>Figure 3:</b>
+
−
                    Representation of the relative fluorescence intensieties of mCherry
+
−
                    (red)
+
−
                    and sfGFP (blue) triplicates after an expression time of 6 h. First, a
+
−
                    constant
+
−
                    concentration of AHT was induced, but the concentration of IPTG was
+
−
                    varied
+
−
                    (0.1 mM or 0.5mM). The induction time of IPFG after inducing with AHT
+
−
                    was
+
−
                    changed as shown in the brackets.
+
−
                    <a href="https://2019.igem.org/wiki/images/d/d5/T--TU_Darmstadt--Ratio_of_sfGFPmCherry_after_6_h.png"
+
−
                        target="_blank">View full size image</a>.
+
−
                </p>
+
−
            </div>
+
−
            <br></br>
+
−
            <p>
+
−
                The next samples were collected after continuing the expression overnight (fig. 4).
+
−
                These samples showed the same trend as the ones taken after 6 h of
+
−
                expression. As before, the difference of the various induction strategies in
+
−
                which IPTG was used showed no significant difference and the uninduced
+
−
                control
+
−
                had the largest excess of sfGFP in this series as well. Unlike the other
+
−
                samples, the AHT only induced variant showed a 1:1 ratio of sfGFP to
+
−
                mCherry.
+
−
            </p>
+
−
            <br></br>
+
−
            <img class="img-fluid center"
+
−
                src="https://2019.igem.org/wiki/images/4/41/T--TU_Darmstadt--Overnight_ratio_of_sfGFPmCherry.png"
+
−
                style="max-width:50%" />
+
−
 
+
−
            <div class="caption">
+
−
                <p>
+
−
                    <b>Figure 4:</b>
+
−
                    Representation of the relative fluorescence intensieties of mCherry
+
−
                    (red)
+
−
                    and sfGFP (blue) after an overnight expression. First, a constant
+
−
                    concentration of AHT was induced, but the concentration of IPTG was
+
−
                    varied
+
−
                    (0.1 mM or 0.5mM). The induction time of IPFG after inducing with AHT
+
−
                    was
+
−
                    changed as shown in the brackets.
+
−
                    <a href="https://2019.igem.org/wiki/images/4/41/T--TU_Darmstadt--Overnight_ratio_of_sfGFPmCherry.png"
+
−
                        target="_blank">View full size image</a>.
+
−
                </p>
+
−
            </div>
+
−
            <br></br>
+
−
            <p>
+
−
                Resulting, the data showed that the T7 site had under any induced condition
+
−
                much
+
−
                a higher activity than the tetA site. The expression was to strong for an
+
−
                effective tuning of the expression levels while IPTG was induced.
+
−
                Surprisingly,
+
−
                the background expression of the T7 site compensated the AHT inducted
+
−
                expression
+
−
                of the tetA site over night and was even stronger at 6 h after induction.
+
−
            </p>
+
−
            <br></br>
+
−
 
+
−
            <h4>Tuning the expression ratio</h4>
+
−
 
+
−
 
+
−
            <p>
+
−
                The spectrophotometric measurements (fig. 5 and 6) of AHT induced trplicates showed a decline in the
+
−
                production of sfGFP with an increasing AHT concentration while the mCherry
+
−
                production seemed relatively constant at all tested concentration.
+
−
                These results were unexpected but provided us with a way to produce varying
+
−
                ratios of sfGFP to mCherry in dependence to the AHT concentration.
+
−
            </p>
+
−
            <br></br>
+
−
            <div class="row">
+
−
 
+
−
                <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
+
−
                    <img class="img-fluid center"
+
−
                        src="https://2019.igem.org/wiki/images/f/f5/T--TU_Darmstadt--TECAN_Dual-Expression-mCherry.png"
+
−
                        style="max-width:50%">
+
−
                    <div class="caption">
+
−
                         <p>
+
−
                            <b>Figure 5:</b>
+
−
                            Spectrophotometric measurement of triplicates with mCherry as
+
−
                            reporter. The
+
−
                            induction of various AHT concentrations was at minute 60. The
+
−
                            induction
+
−
                            concentration ranged from 0.1 - 0.3 µg/mL in steps of 0.5 µg/mL.
+
−
                            In
+
−
                            addition, an uninduced triplicate was observed.
+
−
                            <a href="https://2019.igem.org/wiki/images/f/f5/T--TU_Darmstadt--TECAN_Dual-Expression-mCherry.png"
+
−
                                target="_blank">View full size image</a>.
+
−
                        </p>
+
−
                    </div>
+
−
                </div>
+
−
                <br></br>
+
−
 
+
−
                <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
+
−
                    <img class="img-fluid center"
+
−
                        src="https://2019.igem.org/wiki/images/3/38/T--TU_Darmstadt--TECAN_Dual-Expression-sfGFP.png"
+
−
                        style="max-width:50%">
+
−
                    <div class="caption">
+
−
                        <p>
+
−
                            <b>Figure 6:</b>
+
−
                            Spectrophotometric measurement of triplicates with sfGFP as
+
−
                            reporter. The
+
−
                            induction of various AHT concentrations was at minute 60. The
+
−
                            induction
+
−
                            concentration ranged from 0.1 - 0.3 µg/mL in steps of 0.5 µg/mL.
+
−
                            In
+
−
                            addition, an uninduced triplicate was observed.
+
−
                            <a href="https://2019.igem.org/wiki/images/3/38/T--TU_Darmstadt--TECAN_Dual-Expression-sfGFP.png"
+
−
                                target="_blank">View full size image</a>.
+
−
                        </p>
+
−
                    </div>
+
−
 
+
−
                </div>
+
−
            </div>
+
−
            <br></br>
+
−
            <p>
+
−
                As can be seen in fig. 7, inducer concentrations of AHT ranging
+
−
                from 0.1 µg/mL to 0.4 µg/mL caused a change in the ratio of mCherry:sfGFP
+
−
                from
+
−
                1:2 to 2:1
+
−
                for overnight cultures of <i>E. coli</i>.
+
−
                The ratio seems to approach a maximum of around 2.2:1 following an
+
−
                increasing
+
−
                inducer concentration.
+
−
            </p>
+
−
            <br></br>
+
−
            <img class="img-fluid center"
+
−
                src="https://2019.igem.org/wiki/images/archive/6/6c/20191016075106%21T--TU_Darmstadt--DualRegression-ModRatio.png"
+
−
                style="max-width:50%" />
+
−
            <div class="caption">
+
−
                <p>
+
−
                     <b>Figure 7:</b>
+
−
                    Representation of an asymetric sigmoidal regression (red) of the ratio
+
−
                    of mCherry to sfGFP by various induction concentration of AHT (blue).
+
−
                    The samples were taken in triplicates after an overnight expression. The
+
−
                    function of the regression is shown in equation 1.
+
−
                    <a href="https://2019.igem.org/wiki/images/archive/6/6c/20191016075106%21T--TU_Darmstadt--DualRegression-ModRatio.png"
+
−
                        target="_blank">View full size image</a>.
+
−
                </p>
+
−
            </div>
+
−
            <br></br>
+
−
            <p>
+
−
                After the plotting of the collected data, we did an software-based
+
−
                regression by testing different types functions. The best fitting function
+
−
                with the highest determination coefficient was an asymetric sigmoidal
+
−
                function as presented in equation 1. The determination coefficent of this
+
−
                function is 0.989 which shows the great reliability of this function for
+
−
                further applications.
+
−
            </p>
+
−
            <br></br>
+
−
            <center>
+
−
                <p>
+
−
                    $$y = {0.4756 + {2.5966 \over (1 + 10^{((0.3047 - x) *
+
−
                    11.33)})^{0.2072}}}$$
+
−
                </p>
+
−
            </center>
+
−
            <div class="caption">
+
−
                <p>
+
−
                    <b>Equation 1:</b>
+
−
                    Equation of the asymetric sigmoidal regression with a
+
−
                    determination coefficient of 0.989. The variable x represents the
+
−
                    induction concentration of AHT in µg/mL and y represents the ratio
+
−
                    of mCherry to sfGFP in RFU.
+
−
                </p>
+
−
            </div>
+
  
 +
      <p>
 +
      The sequence of the translation enhancing 5’-UTR can be divided into the four main features listed below:
 +
      </p>
  
 +
   
 +
 +
      <img
 +
        class="img-fluid center"
 +
        src="https://2019.igem.org/wiki/images/f/f3/T--TU_Darmstadt--EnhancerInColor.png"
 +
        style="max-width:85%"
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      />
  
 +
   
 +
    <br></br>
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      <div class="container-noborders">
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        <div class="table-responsive-sm">
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          <table class="table table-light">
 +
            <tr>
 +
              <th scope="col">Sequence</th>
 +
              <th scope="col">Function</th>
 +
            </tr>
 +
            <tr>
 +
              <td style="color:#d59b9b; word-wrap:break-word;">
 +
                AATAATTTTGTT<br />TTAACTTTAA
 +
              </td>
 +
              <td>
 +
              The T7 g10 leader sequence (first described by Olins <i>et</i> al<sup id="cite_ref-1" class="reference">
 +
                        <a href="#cite_note-1">[1] </a>
 +
                    </sup>)increases the efficiency of translation initiation. This sequence contains the epsilon motif TTAACTTTA which enhances the binding of the mRNA to the 16S rRNA.
 +
            </td>
 +
            </tr>
 +
            <tr>
 +
              <td style="color:#a5c8c8;">poly-A</td>
 +
              <td>
 +
                Referring to Takahashi et al.<sup id="cite_ref-2" class="reference">
 +
                        <a href="#cite_note-2">[2] </a>
 +
                    </sup> a spacer between the epsilon motive and the RBS improves the translation rate.
 +
              </td>
 +
            </tr>
 +
            <tr>
 +
              <td style="color:#698888;">GAAGGAG</td>
 +
              <td>
 +
              According to Karig <i>et</i> al.<sup id="cite_ref-3" class="reference">
 +
                        <a href="#cite_note-3">[3] </a>
 +
                    </sup> and Lentini et. al<sup id="cite_ref-4" class="reference">
 +
                        <a href="#cite_note-4">[4] </a> </sup> a distance of 4-9 bases between RBS and start codon increases the translation efficiency.
 +
              </td>
 +
            </tr>
 +
            <tr>
 +
              <td style="color:#cc9966;">AATAATCT</td>
 +
              <td>
 +
                According to Lentini et. al<sup id="cite_ref-4" class="reference">
 +
                        <a href="#cite_note-4">[4] </a> </sup> an AT-rich composition between the RBS and the start codon results in the best expression results.
 +
              </td>
 +
            </tr>
 +
          </table>
 +
        </div>
 +
      </div>
 +
       
 +
            <h3>Results</h3>
 +
            <hr class="head">
  
 +
   
 +
      <p>
 +
  Fig. 1 shows the original version of the part (left) and the improved version (right) after overnight cultivation on an agar plate and after cultivation for 24 h in M9 minimal medium for better visualization of the emitted light at 512 nm<sup id="cite_ref-5" class="reference">
 +
                        <a href="#cite_note-5">[5] </a> </sup>. Both the agar plate and the liquid culture were supplemented with chloramphenicol according to the cmp resistance on the pSB1C3 backbone. Both pictures were taken using a dark reader (Dark Reader Clare Chemical Research) for better visualization of the yellow color.
 +
      </p>
  
 +
  <img
 +
        class="img-fluid center"
 +
        src="https://2019.igem.org/wiki/images/0/0a/T--TU_Darmstadt--amilGFPComparison.png"
 +
        style="max-width:90%"
 +
      />
 +
<div class="caption">
 +
                                                        <p>
 +
                                                            <b>
 +
                                                                <center> Figure 2:
 +
    </b><i>E. coli</i> BL21 DE3 transformed with BBa_K1073024 after cultivation on LB-agar (left) and in a liquid culture (M9 minimal medium) supplemented with cmp. The improved version of the part shows increased amilGFP expression </center>
 +
                                                        </p>
 +
                    </div>
 +
<p>
 +
Both the agar plate and the liquid culture show an enhanced expression of the amilGFP gene after the upstream insertion of the translation enhancing 5’-UTR.
 +
</p>
  
 
</html>
 
</html>

Revision as of 19:33, 18 October 2019


Constitutively expressed chromoprotein amilGFP with Translation Enhancing 5'-UTR

Improved Version of BBa_K1073024

Usage and Biology

BBa_K3187015 (improved BBa_K1073024) encodes the yellow chromoprotein amilGFP derived from the coral Acropora millepora [5] under the control of a strong constitutive promoter (BBa_J23100) in combination with a ribosomal binding site ( BBa_B0032). The improved and the original version of the part were cloned into the pSB1C3 backbone and transformed in E. coli BL21 (DE3).

Both parts were improved in terms of their expression levels, based on the insertion of a 5’ untranslated region (5’-UTR) upstream of the coding sequence. This 5'-UTR was adapted from iGEM Bielefeld 2015 (  BBa_K1758100) and is based on the research of Olins et al [1] and Takahashi et al. [2] . It contains the strong ribosomal binding site (RBS) g10-L from the T7 bacteriophage and a sequence that plays a role in the regulation of mRNA binding to and release from the 30S ribosomal subunit. The 5'-UTR therefore enhances the translation efficiency of the following coding sequence (CDS) (Fig. 1).

Figure 1: Schematic depiction of the composition and interaction of the enhancer sequence with the 30S ribosomal subunit described by Takahashi et. al. [2] .

The sequence of the translation enhancing 5’-UTR can be divided into the four main features listed below:



Sequence Function
AATAATTTTGTT
TTAACTTTAA 		The T7 g10 leader sequence (first described by Olins et al [1] )increases the efficiency of translation initiation. This sequence contains the epsilon motif TTAACTTTA which enhances the binding of the mRNA to the 16S rRNA.
poly-A Referring to Takahashi et al. [2] a spacer between the epsilon motive and the RBS improves the translation rate.
GAAGGAG According to Karig et al. [3] and Lentini et. al [4] a distance of 4-9 bases between RBS and start codon increases the translation efficiency.
AATAATCT According to Lentini et. al [4] an AT-rich composition between the RBS and the start codon results in the best expression results.

Results

Fig. 1 shows the original version of the part (left) and the improved version (right) after overnight cultivation on an agar plate and after cultivation for 24 h in M9 minimal medium for better visualization of the emitted light at 512 nm [5] . Both the agar plate and the liquid culture were supplemented with chloramphenicol according to the cmp resistance on the pSB1C3 backbone. Both pictures were taken using a dark reader (Dark Reader Clare Chemical Research) for better visualization of the yellow color.

Figure 2: E. coli BL21 DE3 transformed with BBa_K1073024 after cultivation on LB-agar (left) and in a liquid culture (M9 minimal medium) supplemented with cmp. The improved version of the part shows increased amilGFP expression

Both the agar plate and the liquid culture show an enhanced expression of the amilGFP gene after the upstream insertion of the translation enhancing 5’-UTR.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]