Difference between revisions of "Part:BBa K3187011"
 
(13 intermediate revisions by the same user not shown)
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<html>
 
<html>
 +
<script id="MathJax-script" async
 +
    src="https://2019.igem.org/wiki/index.php?title=Template:TU_Darmstadt/MathjaxJS&action=raw&ctype=text/javascript"></script>
  
 
<div class="container">
 
<div class="container">
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             <h3>Profile</h3>
 
             <h3>Profile</h3>
 +
            <hr class="head">
 +
 
             <table style=“width:80%“>
 
             <table style=“width:80%“>
 
                 <tr>
 
                 <tr>
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                 </tr>
 
                 </tr>
 
             </table>
 
             </table>
−
 
+
            <br></br>
 
             <h3>Usage and Biology</h3>
 
             <h3>Usage and Biology</h3>
 +
            <hr class="head">
 +
 
             <p>
 
             <p>
 
                 This composite part is a dual expression plasmid with pTeTW3con2 as backbone. It was cloned to
 
                 This composite part is a dual expression plasmid with pTeTW3con2 as backbone. It was cloned to
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                 The fluorescence of mCherry and sfGFP can be measured and acts as expression reporters.
 
                 The fluorescence of mCherry and sfGFP can be measured and acts as expression reporters.
 
             </p>
 
             </p>
−
 
+
            <br></br>
  
 
             <h3>Results</h3>
 
             <h3>Results</h3>
 +
            <hr class="head">
  
 
             <h4>Cloning</h4>
 
             <h4>Cloning</h4>
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                 checked via sequencing.
 
                 checked via sequencing.
 
             </p>
 
             </p>
−
 
+
            <br></br>
 
             <h4>Measuring the expression levels after single induction</h4>
 
             <h4>Measuring the expression levels after single induction</h4>
  
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                 of the instrument while a change in the signal of mCherry was barely
 
                 of the instrument while a change in the signal of mCherry was barely
 
                 detected
 
                 detected
−
                 during the measuring process. For this reason, we changed our method to an
+
                 during the measuring process.
−
                expression assay where the fluorescence was detected after a semi
+
−
                denaturized
+
−
                SDS PAGE via a fluorescence imager.
+
 
             </p>
 
             </p>
  
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                 <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
 
                 <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
−
                     <img class="img-fluid center"
+
                     <center>
−
                        src="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Spectramax_AHT_%28Min_Max%29.png"
+
                        <img class="img-fluid center"
−
                        style="max-width:50%">
+
                            src="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Spectramax_AHT_%28Min_Max%29.png"
−
                    <div class="caption">
+
                            style="max-width:50%">
−
                        <p>
+
                        <div class="caption">
−
                            <b>Figure 1:</b>
+
                            <p>
−
                            Spectrophotometric measurement of the fluorescences of mCherry
+
                                <b>Figure 1:</b>
−
                            (red) and sfGFP (blue) triplicates after inducing with AHT. AHT
+
                                Spectrophotometric measurement of the fluorescences of mCherry
−
                            was induced at 90 minutes. The iduction with 0.1 µg/mL is shown
+
                                (red) and sfGFP (blue) triplicates after inducing with AHT. AHT
−
                            in light red and blue and 0.3 µg/mL is shown in dark red and
+
                                was induced at 90 minutes. The iduction with 0.1 µg/mL is shown
−
                            blue.
+
                                in light red and blue and 0.3 µg/mL is shown in dark red and
−
                            <a href="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Spectramax_AHT_%28Min_Max%29.png"
+
                                blue.
−
                                target="_blank">View full size image</a>.
+
                                <a href="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Spectramax_AHT_%28Min_Max%29.png"
−
                        </p>
+
                                    target="_blank">View full size image</a>.
−
                     </div>
+
                            </p>
 +
                     </center>
 
                 </div>
 
                 </div>
−
 
+
            </div>
−
                <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
+
            <br></br>
 +
            <div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
 +
                <center>
 
                     <img class="img-fluid center"
 
                     <img class="img-fluid center"
 
                         src="https://2019.igem.org/wiki/images/7/7c/T--TU_Darmstadt--Spectramax_IPTG_%28Min_Max%29.png"
 
                         src="https://2019.igem.org/wiki/images/7/7c/T--TU_Darmstadt--Spectramax_IPTG_%28Min_Max%29.png"
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                                 target="_blank">View full size image</a>.
 
                                 target="_blank">View full size image</a>.
 
                         </p>
 
                         </p>
−
                    </div>
+
                 </center>
−
 
+
−
                 </div>
+
 
             </div>
 
             </div>
  
−
            <h4>Testing various dual induction strategies</h4>
+
        </div>
 +
    </div>
 +
    <br></br>
 +
    <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>
 +
    <center>
 +
        <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>
 
             <p>
−
                 The results of the samples which were collected after 6 h of expression (fig. 3)
+
                 <b>Figure 3:</b>
−
                 showed a
+
                Representation of the relative fluorescence intensieties of mCherry
−
                 distinct trend. Whenever IPTG was induced during the experimental procedure
+
                (red)
−
                there was a large excess of sfGFP in comparison to mCherry. The induction
+
                 and sfGFP (blue) triplicates after an expression time of 6 h. First, a
−
                 times
+
                 constant
−
                 or used concentrations of IPTG just had a small effect on the resulting
+
                concentration of AHT was induced, but the concentration of IPTG was
−
                 ratio of
+
                 varied
−
                 sfGFP to mCherry. Only the variant which was just induced with AHT showed a
+
                 (0.1 mM or 0.5mM). The induction time of IPFG after inducing with AHT
−
                 significant difference having a 2:1 ratio of sfGFP to mCherry. As expected
+
                 was
−
                from
+
                 changed as shown in the brackets.
−
                the previous experiments, the uninduced control showed the highest excess of
+
                 <a href="https://2019.igem.org/wiki/images/d/d5/T--TU_Darmstadt--Ratio_of_sfGFPmCherry_after_6_h.png"
−
                sfGFP in this series.
+
                    target="_blank">View full size image</a>.
 
             </p>
 
             </p>
 +
    </center>
 +
</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>
 +
<center>
 +
    <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%" />
  
−
            <img class="img-fluid center"
+
    <div class="caption">
−
                src="https://2019.igem.org/wiki/images/d/d5/T--TU_Darmstadt--Ratio_of_sfGFPmCherry_after_6_h.png"
+
        <p>
−
                 style="max-width:50%" />
+
            <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>
 +
</center>
 +
</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>
  
−
            <div class="caption">
+
<h4>Tuning the expression ratio</h4>
−
                <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>
+
  
−
            <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>
 
  
 +
<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 ">
 +
        <center>
 
             <img class="img-fluid center"
 
             <img class="img-fluid center"
−
                 src="https://2019.igem.org/wiki/images/4/41/T--TU_Darmstadt--Overnight_ratio_of_sfGFPmCherry.png"
+
                 src="https://2019.igem.org/wiki/images/f/f5/T--TU_Darmstadt--TECAN_Dual-Expression-mCherry.png"
−
                 style="max-width:50%" />
+
                 style="max-width:50%">
−
 
+
 
             <div class="caption">
 
             <div class="caption">
 
                 <p>
 
                 <p>
−
                     <b>Figure 4:</b>
+
                     <b>Figure 5:</b>
−
                     Representation of the relative fluorescence intensieties of mCherry
+
                     Spectrophotometric measurement of triplicates with mCherry as
−
                     (red)
+
                     reporter. The
−
                    and sfGFP (blue) after an overnight expression. First, a constant
+
                     induction of various AHT concentrations was at minute 60. The
−
                     concentration of AHT was induced, but the concentration of IPTG was
+
                     induction
−
                     varied
+
                     concentration ranged from 0.1 - 0.3 µg/mL in steps of 0.5 µg/mL.
−
                     (0.1 mM or 0.5mM). The induction time of IPFG after inducing with AHT
+
                     In
−
                     was
+
                     addition, an uninduced triplicate was observed.
−
                     changed as shown in the brackets.
+
                     <a href="https://2019.igem.org/wiki/images/f/f5/T--TU_Darmstadt--TECAN_Dual-Expression-mCherry.png"
−
                     <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>.
 
                         target="_blank">View full size image</a>.
 
                 </p>
 
                 </p>
−
            </div>
+
        </center>
 +
    </div>
 +
</div>
 +
<br></br>
  
 +
<div class="col-12 col-sm-12 col-md-12 col-xl-6 my-3 ">
 +
    <center>
 +
        <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>
 
             <p>
−
                 Resulting, the data showed that the T7 site had under any induced condition
+
                 <b>Figure 6:</b>
−
                 much
+
                 Spectrophotometric measurement of triplicates with sfGFP as
−
                 a higher activity than the tetA site. The expression was to strong for an
+
                 reporter. The
−
                 effective tuning of the expression levels while IPTG was induced.
+
                 induction of various AHT concentrations was at minute 60. The
−
                 Surprisingly,
+
                 induction
−
                 the background expression of the T7 site compensated the AHT inducted
+
                 concentration ranged from 0.1 - 0.3 µg/mL in steps of 0.5 µg/mL.
−
                 expression
+
                 In
−
                 of the tetA site over night and was even stronger at 6 h after induction.
+
                 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>
 
             </p>
 +
    </center>
 +
</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>
 +
<center>
 +
    <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>
 +
</center>
 +
<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>
 +
<br></br>
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Latest revision as of 11:14, 20 October 2019

pT7 Superfolder Green Fluorescence Protein x pTet mCherry (Convergent)

Profile

Name pTeTW3con2-ptet-mCherry--sfGFP-pT7
Base pairs 1780
Molecular weight 26.7 kDa (mCherry) + 26.8 kDa (sfGFP)
Origin Synthetic
Parts tetA promoter, T7 promoter, T7 terminator, Lac Operator, RBS, mCherry, sfGFP
Properties Dual expression of mCherry and sfGFP as reporters to monitor expression levels of the tetA and T7 site.


Usage and Biology

This composite part is a dual expression plasmid with pTeTW3con2 as backbone. It was cloned to characterize the expression levels of the two convergent expression sites. The tetA promoter is inducible with anhydrotetracycline (AHT) and the site (BBa_K3187039) encodes the protein mCherry. The T7 promoter can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the site (BBa_K3187040) encodes the protein sfGFP.

The fluorescence of mCherry and sfGFP can be measured and acts as expression reporters.



Results

Cloning

pTeTW3con2-ptet-mCherry--sfGFP-pT7 was cloned in two steps via a restriction and ligation protocol. First, the mCherry gene was cloned into the backbone pTeTW3con2. Sequencing analysis was carried out to test whether the cloning was positive before the next step started. Next, the sfGFP gene was cloned into the backbone (pTeTw3con2-ptet-mCherry). The cloning fo the final product was checked via sequencing.



Measuring the expression levels after single induction

The measurement (fig. 1 and 2) showed a strong background expression of the T7 site represented by the increasing fluorescence signal of sfGFP in the uninduced condition. 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.

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 of the instrument while a change in the signal of mCherry was barely detected during the measuring process.

Figure 1: 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. View full size image.



Figure 2: Spectrophotometric measurement of the fluorescences of mCherry (red) and sfGFP (blue) triplicates after inducing with IPTG. IPTG was induced at 90 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. View full size image.



Testing various dual induction strategies

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.



Figure 3: 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. View full size image.



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.



Figure 4: 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. View full size image.



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.



Tuning the expression ratio

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.



Figure 5: 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. View full size image.



Figure 6: 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. View full size image.



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 E. coli. The ratio seems to approach a maximum of around 2.2:1 following an increasing inducer concentration.



Figure 7: 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. View full size image.



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.



$$y = {0.4756 + {2.5966 \over (1 + 10^{((0.3047 - x) * 11.33)})^{0.2072}}}$$

Equation 1: 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.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 1729
    Illegal PstI site found at 805
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 76
    Illegal NheI site found at 810
    Illegal PstI site found at 805
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 76
    Illegal XhoI site found at 970
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 1729
    Illegal PstI site found at 805
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 1729
    Illegal PstI site found at 805
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1674