Difference between revisions of "Part:BBa K3187011"
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             <h4>Measuring the expression levels after single induction</h4>
 
             <h4>Measuring the expression levels after single induction</h4>
 +
  
 
             <p>
 
             <p>
 +
                The measurement (fig. 1 and 2) showed a strong background expression of the T7 site
 +
                represented
 +
                by the increasing fluorescence signal of sfGFP.
 +
                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
 +
                of the instrument while a change in the signal of mCherry was barely
 +
                detected
 +
                during the measuring process. For this reason, we changed our method to an
 +
                expression assay where the fluorescence was detected after a semi
 +
                denaturized
 +
                SDS PAGE via a fluorescence imager.
 
             </p>
 
             </p>
 +
 +
            <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/f4/T--TU_Darmstadt--Spectramax_AHT_%28Min_Max%29.png"
 +
                        style="max-width:100%">
 +
                    <div class="caption">
 +
                        <p>
 +
                            <b>Figure 1:</b>
 +
                            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>.
 +
                        </p>
 +
                    </div>
 +
                </div>
 +
 +
                <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/7/7c/T--TU_Darmstadt--Spectramax_IPTG_%28Min_Max%29.png"
 +
                        style="max-width:100%">
 +
                    <div class="caption">
 +
                        <p>
 +
                            <b>Figure 2:</b>
 +
                            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.
 +
                            <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>.
 +
                        </p>
 +
                    </div>
 +
 +
                </div>
 +
            </div>
  
 
             <h4>Testing various dual induction strategies</h4>
 
             <h4>Testing various dual induction strategies</h4>
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             <p>
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             </p>
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</html>
 
</html>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 13:08, 18 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. 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. For this reason, we changed our method to an expression assay where the fluorescence was detected after a semi denaturized SDS PAGE via a fluorescence imager.

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

Tuning the expression ratio


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