Difference between revisions of "Part:BBa K2144008"
 
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<p>We purified the Sortase A from team Stockholm via fast protein liquid chromatography (FPLC) using the ÄKTA pure (fig.
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<p>At the start of our project, we looked for a Sortase A we could use for our project.  We discovered a matching BioBrick in the registry that was submitted by the iGEM team Stockholm in the year 2016. We added an RBS upstream and a T7 terminator downstream of the coding sequence for the means of expression. We transformed our <i>E. coli</i> cells and started to express this Ca<sup>2+</sup>-dependent Sortase A. For comparison, we expressed Sortase A7M and Sortase A5M additional to Sortase A from Stockholm. As usual, we purified the Sortase A BBa_K2144008 via fast protein liquid chromatography (FPLC) using the ÄKTA pure <b>(Fig. 1)</b> by means of His-tag purification. </p><br>
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    2). In order to purify the Sortase A from team Stockholm we used the existing His-tag.</p>
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< ----- Bild von ÄKTA Chromatogram ---->
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<center>
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<div class="row py-3">
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                <div class="col-xs-12 col-sm-12 col-md-6 py-2 order-md-1">
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<img class="img-fluid center"
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        src="https://2019.igem.org/wiki/images/c/c5/T--TU_Darmstadt--Chrom_SrtASandereProteine-1734-.png"
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        style=max-width:60%;>
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    <div class="caption">
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        <p>
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            <b>
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                Figure 1:
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            </b>Chromatogram of the ÄKTA run. The elution of other proteins than Sortase A is shown.
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        </p>
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    </div>
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</div>
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<div class="col-xs-12 col-sm-12 col-md-6 py-2 order-md-2">
  
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    <p>Fig 1: Chromatogram of the ÄKTA during the purification of Sortase A from team Stockholm.
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<img class="img-fluid center"
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        In figure 2 it is shown that the Sortase A from Stockholm eluted at ???. This should make sure that we have
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         src="https://2019.igem.org/wiki/images/d/db/T--TU_Darmstadt--Chrom_SrtAS-PeakElution-1735-.png"
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        purified the right protein with the fitting tag. <br>
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         style=max-width:60%;>
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        We then confirmed the data of the purification by checking on the size of the Sortase using SDS-PAGE (fig. 2).
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        We purified it twice with the same result in size.
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    </p>
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    <br><br>
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    <img class="img-fluid center"
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         src="https://2019.igem.org/wiki/images/archive/b/b4/20191018135731%21T--TU_Darmstadt--Klara_Sortase_Stockholm_Gr%C3%B6%C3%9Fe.png"
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         style=max-width:80%;>
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     <div class="caption">
 
     <div class="caption">
 
         <p>
 
         <p>
 
             <b>
 
             <b>
 
                 Figure 2:
 
                 Figure 2:
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             </b>A SDS-PAGE of a triplicate of the reaction solution for the sortase reaction consisting of coat protein
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             </b>Chromatogram of the ÄKTA run. Sortase A BBa_K2144008 eluted as a peak at about 59&nbsp;mL.
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            (CP), mCherry and Sortase A from Stockholm which were incubated for 90 minutes at 37 °C. A negative control
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            without Sortase A from Stockholm in a triplicate is also included. To verify the size the Sortase A from
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            Stockholm was also put as a control on the gel solely.
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         </p>
 
         </p>
 
     </div>
 
     </div>
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</div>
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</div>
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  <br> <p>In <b>Fig.1</b> it is shown that the Sortase A BBa_K2144008 eluted at about 59&nbsp;mL.<b>Fig. 2</b> shows the elution of other proteins than the Sortase A. Expected size and purity of the protein were assessed by SDS-PAGE <b>(Fig.3)</b>
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    </p>
 
     <br><br>
 
     <br><br>
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    <p>In figure 2 a SDS-PAGE of the first sortase reaction performed with the Sortase A from Stockholm is shown as
 
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        well. On the right is a triplicate of the purified Sortase A from Stockholm to reassure it has the right size of
 
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        about 15 kDa which is the case. On the left are triplicates of coat protein with an LPETGG-tag (CP-LPETGG) and
 
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        GGGG-mCherry. The first three are with Sortase Stockholm in the reaction and the middle three are without
 
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        Sortase Stockholm. The reaction was run at 37 °C for 90 minutes. Seeing this first gel arose the suspicion that
 
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        the Sortase A from team Stockholm is not working properly. We then tried to confirm whether our suspicion was
 
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        right. In order to do so we ran another test with a SDS-PAGE (fig. 3) and additionally used the Sortase A from
 
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        Stockholm for one of our self-made FRET reactions. <br><br> </p>
 
 
     <img class="img-fluid center"
 
     <img class="img-fluid center"
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         src="https://2019.igem.org/wiki/images/c/c1/T--TU_Darmstadt--Klara_Sortase_Stockholm_Funktion.png"
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         src="https://2019.igem.org/wiki/images/archive/b/b4/20191019083449%21T--TU_Darmstadt--Klara_Sortase_Stockholm_Gr%C3%B6%C3%9Fe.png"
 
         style=max-width:80%;>
 
         style=max-width:80%;>
 
     <div class="caption">
 
     <div class="caption">
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             <b>
 
             <b>
 
                 Figure 3:
 
                 Figure 3:
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             </b>A SDS-PAGE of the sortase reaction connecting CP-LPETGG and GGGG-mCherry. The gel includes a positive
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             </b>A SDS-PAGE with increased contrast of a triplicate of the reaction solution for the sortase reaction consisting of coat protein (CP), mCherry and Sortase A from Stockholm which were incubated for 90 minutes at 37 °C. A negative control without Sortase A from Stockholm in a triplicate is also included. To verify the size the Sortase A from Stockholm was also put as a control on the gel solely.  
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            triplicate consisting of CP-LPETGG, GGGG-mCherry and Sortase A from Stockholm and two negative controls each
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            in a triplicate of which one is similar to the positive control but without 10 mM calcium in the reaction
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            buffer and one is without Sortase A in the reaction.
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        </p>
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     </div>
 
     </div>
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     <br><br>
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     <br>
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     <p>The gel in figure 3 shows a first proof for our suspicion. The first triplicate on the left contains CP-LPETGG,
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</center>
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        GGGG-mCherry and Sortase from Stockholm in a buffer with 10 mM calcium present. The middle triplicate shows
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     <p>In <b>Fig.3</b> a SDS-PAGE of the first sortase reaction performed with the Sortase A from Stockholm is shown as well. On the right is a triplicate of the purified Sortase A BBa_K2144008 to reassure it has the right size of about 15 kDa which is the case. The solvent front appears below the Sortase A BBa_K2144008 band. CP-LPETGG and GGGG-mCherry were mixed and Sortase A BBa_K2144008 was added. The reaction was run for 90 minutes at 37 °C. As a negative control, Sortase was omitted solely. The difference in the visibility of the bands of Sortase BBa_K2144008 in the positive and negative control resulted from the positive control being diluted 1:2 after the reaction. The other negative control includes CP-LPETGG and GGGG-mCHerry without the Sortase BBa_K2144008 added. As we expected to see a band at about 74 kDa in the positive control we suspected that the Sortase A BBa_K2144008 is not working properly. We then tried to confirm whether our suspicion was right by performing a FRET-assay.
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        CP-LPETGG, GGGG-mCherry and Sortase from Stockholm but in a buffer with no calcium present. The last triplicate
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<br>In order to have a final proof we compared our Sortase A7M to the Sortase A BBa_K2144008 using a FRET-assay connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP <b>(Fig. 4)</b>. We measured the reaction kinetics at 30 °C.<br></p>
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        does only contain CP-LPETGG and GGGG-mCherry but no Sortase A from Stockholm. The reaction was performed at 37
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<center>
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        °C for 90 minutes. As all three sample show the same outcome it can be assumend that the Sortase A from team
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        Stockholm does not show any activity whatsoever.<br>
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        In order to have a final proof we compared our Sortase A7M to the Sortase A from Stockholm using a FRET
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        connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP (fig. 4). We ran this
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        reaction for 3h at 30 °C.<br></p>
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     <img class="img-fluid center"
 
     <img class="img-fluid center"
 
         src="https://2019.igem.org/wiki/images/6/64/T--TU_Darmstadt--mCherry_GFP_FRET_different_sortases.png"
 
         src="https://2019.igem.org/wiki/images/6/64/T--TU_Darmstadt--mCherry_GFP_FRET_different_sortases.png"
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                 Figure 4:
 
                 Figure 4:
 
             </b>A FRET-assay of Sortase A from Stockholm and Sortase A7M connecting TAMRA-LPETG and GGGG-mCherry with
 
             </b>A FRET-assay of Sortase A from Stockholm and Sortase A7M connecting TAMRA-LPETG and GGGG-mCherry with
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             and without 10mM calcium. The ΔRFU refers to the respective negeative control without Sortase.
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             and without 10mM calcium. The ΔRFU refers to the respective negative control without each sortase.
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.
 
         </p>
 
         </p>
 
     </div>
 
     </div>
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    <br><br>
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</center>
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     <p>As visible in figure 4 the Sortase A from Stockholm does not show any activity during the reaction although it
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    <br>
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        was incubated with 10mM calcium present. In contrary to our Sortase A7M which was incubated without calcium
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     <p>As visible in <b>Fig.4</b> the Sortase A BBa_K2144008 does not show any activity during the reaction although 10mM calcium was present in the reaction buffer. In contrary, the Sortase A7M, incubated without calcium, shows the expected increase in fluorescence visible in the ΔRFU at 514 nm over time. The ΔRFU refers to the difference between the negative control without the respective sortase. In comparison the Sortase A BBa_K2144008  does not seem to catalyze any reactions over the measured time span. If there would be any activity it would look similar to the graph of the Sortase A7M where the ΔRFU at 514 nm is increased due to the FRET pair being connected. This confirms the SDS-PAGE results We have to conclude that Sortase A BBa_K2144008 is not functional.<br></p>
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        because it is an independent mutant. The Sortase A7M also shows the expected increase in fluorescence visible in
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        the ΔRFU at 514 nm over time. The ΔRFU refers to the difference between the negative control without the
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        respective sortase (data not shown). In comparison the Stockholm Sortase A does not seem to catalyze any
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        reactions over the measured time span. If there would be any activity it would look similar to the graph of the
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        Sortase A7M where the ΔRFU at 514 nm is increased due to the FRET pair being connected. This confirms the
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        SDS-PAGEs that showed the same outcome of the Sortase A from Stockholm being not functional independent from the
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        presence of calcium.<br><br></p>
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Latest revision as of 20:11, 20 October 2019

Coding sequence for Sortase A with His-tag

Usage

Sortase A is a bacterial enzyme with the ability to break and form new peptide bonds. The key feature of the enzyme is the specific conjugation reaction it carries out, where the enzyme recognizes a specific amino acid sequence, a so called sorting motif (LPXTG motif in the case of S.aureus) and conjugate this sequence with another unit carrying an oligo glycine motif where a new peptide bond is formed [1],[2].

Biology & BioBrick Design

This Biobrick is a truncated version of the enzyme where the transmembrane domain (amino acids 1-59) is not included in the coding sequence to increase solubility [2]. Further, the BioBrick has also been fused with the Protein G B1 Domain (GB1), upstream the Sortase coding sequence, acting as a solubility tag and a His-tag to enabling purification through IMAC [2].

Characterization by iGEM TU_Darmstadt 2019 (Expression and assays)

At the start of our project, we looked for a Sortase A we could use for our project. We discovered a matching BioBrick in the registry that was submitted by the iGEM team Stockholm in the year 2016. We added an RBS upstream and a T7 terminator downstream of the coding sequence for the means of expression. We transformed our E. coli cells and started to express this Ca2+-dependent Sortase A. For comparison, we expressed Sortase A7M and Sortase A5M additional to Sortase A from Stockholm. As usual, we purified the Sortase A BBa_K2144008 via fast protein liquid chromatography (FPLC) using the ÄKTA pure (Fig. 1) by means of His-tag purification.


Figure 1: Chromatogram of the ÄKTA run. The elution of other proteins than Sortase A is shown.

Figure 2: Chromatogram of the ÄKTA run. Sortase A BBa_K2144008 eluted as a peak at about 59 mL.


In Fig.1 it is shown that the Sortase A BBa_K2144008 eluted at about 59 mL.Fig. 2 shows the elution of other proteins than the Sortase A. Expected size and purity of the protein were assessed by SDS-PAGE (Fig.3)



Figure 3: A SDS-PAGE with increased contrast of a triplicate of the reaction solution for the sortase reaction consisting of coat protein (CP), mCherry and Sortase A from Stockholm which were incubated for 90 minutes at 37 °C. A negative control without Sortase A from Stockholm in a triplicate is also included. To verify the size the Sortase A from Stockholm was also put as a control on the gel solely.


In Fig.3 a SDS-PAGE of the first sortase reaction performed with the Sortase A from Stockholm is shown as well. On the right is a triplicate of the purified Sortase A BBa_K2144008 to reassure it has the right size of about 15 kDa which is the case. The solvent front appears below the Sortase A BBa_K2144008 band. CP-LPETGG and GGGG-mCherry were mixed and Sortase A BBa_K2144008 was added. The reaction was run for 90 minutes at 37 °C. As a negative control, Sortase was omitted solely. The difference in the visibility of the bands of Sortase BBa_K2144008 in the positive and negative control resulted from the positive control being diluted 1:2 after the reaction. The other negative control includes CP-LPETGG and GGGG-mCHerry without the Sortase BBa_K2144008 added. As we expected to see a band at about 74 kDa in the positive control we suspected that the Sortase A BBa_K2144008 is not working properly. We then tried to confirm whether our suspicion was right by performing a FRET-assay.
In order to have a final proof we compared our Sortase A7M to the Sortase A BBa_K2144008 using a FRET-assay connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP (Fig. 4). We measured the reaction kinetics at 30 °C.

Figure 4: A FRET-assay of Sortase A from Stockholm and Sortase A7M connecting TAMRA-LPETG and GGGG-mCherry with and without 10mM calcium. The ΔRFU refers to the respective negative control without each sortase. .


As visible in Fig.4 the Sortase A BBa_K2144008 does not show any activity during the reaction although 10mM calcium was present in the reaction buffer. In contrary, the Sortase A7M, incubated without calcium, shows the expected increase in fluorescence visible in the ΔRFU at 514 nm over time. The ΔRFU refers to the difference between the negative control without the respective sortase. In comparison the Sortase A BBa_K2144008 does not seem to catalyze any reactions over the measured time span. If there would be any activity it would look similar to the graph of the Sortase A7M where the ΔRFU at 514 nm is increased due to the FRET pair being connected. This confirms the SDS-PAGE results We have to conclude that Sortase A BBa_K2144008 is not functional.


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
    COMPATIBLE WITH RFC[1000]


References

[1] Popp, M. W.-L. and Ploegh, H. L. (2011), Making and Breaking Peptide Bonds: Protein Engineering Using Sortase. Angew. Chem. Int. Ed., 50: 5024–503

[2] Westerlund, K. Karlstrom, A., Honarvar, H., Tolmachev, V. Design, Preparation, and Characterization of PNA-Based Hybridization Probes for Affibody-Molecule-Mediated Pretargeting. Bioconjugate Chem., 2015, 26 (8), pp 1724–1736