Difference between revisions of "Part:BBa K2144008"

(→‎Characterization by iGEM TU_Darmstadt 2019 (Expression and FRET-based assay))
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2144008 short</partinfo>
 
<partinfo>BBa_K2144008 short</partinfo>
  
 
===Usage===
 
===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].
+
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===
 
===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].
+
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 FRET-based assay)===
 
===Characterization by iGEM TU_Darmstadt 2019 (Expression and FRET-based assay)===
  
−
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+
<p>We purified the Sortase A from team Stockholm via fast protein liquid chromatography (FPLC) using the ÄKTA pure (fig.
 +
    2). In order to purify the Sortase A from team Stockholm we used the existing His-tag.</p>
  
−
<!-- -->
+
< ----- Bild von ÄKTA Chromatogram ---->
−
<span class='h3bb'>Sequence and Features</span>
+
 
−
<partinfo>BBa_K2144008 SequenceAndFeatures</partinfo>
+
    <p>Fig 1: Chromatogram of the ÄKTA during the purification of Sortase A from team Stockholm.
 +
        In figure 2 it is shown that the Sortase A from Stockholm eluted at ???. This should make sure that we have
 +
        purified the right protein with the fitting tag. <br>
 +
        We then confirmed the data of the purification by checking on the size of the Sortase using SDS-PAGE (fig. 2).
 +
        We purified it twice with the same result in size.<br><br></p>
 +
    <img class="img-fluid center"
 +
        src="https://2019.igem.org/wiki/images/archive/b/b4/20191018135731%21T--TU_Darmstadt--Klara_Sortase_Stockholm_Gr%C3%B6%C3%9Fe.png"
 +
        style=max-width:80%;>
 +
    <div class="caption">
 +
        <p>
 +
            <b>
 +
                <center> Figure 2:
 +
            </b>A SDS-PAGE 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. </center>
 +
        </p>
 +
    </div>
 +
 
 +
    <p>In figure 2 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 from Stockholm to reassure it has the right size of
 +
        about 15 kDa which is the case. On the left are triplicates of coat protein with an LPETGG-tag (CP-LPETGG) and
 +
        GGGG-mCherry. The first three are with Sortase Stockholm in the reaction and the middle three are without
 +
        Sortase Stockholm. The reaction was run at 37 °C for 90 minutes. Seeing this first gel arose the suspicion that
 +
        the Sortase A from team Stockholm is not working properly. We then tried to confirm whether our suspicion was
 +
        right. In order to do so we ran another test with a SDS-PAGE (fig. 3) and additionally used the Sortase A from
 +
        Stockholm for one of our self-made FRET reactions. <br><br> </p>
 +
    <img class="img-fluid center"
 +
        src="https://2019.igem.org/wiki/images/c/c1/T--TU_Darmstadt--Klara_Sortase_Stockholm_Funktion.png"
 +
        style=max-width:80%;>
 +
    <div class="caption">
 +
        <p>
 +
            <b>
 +
                <center> Figure 3:
 +
            </b>A SDS-PAGE of the sortase reaction connecting CP-LPETGG and GGGG-mCherry. The gel includes a positive
 +
            triplicate consisting of CP-LPETGG, GGGG-mCherry and Sortase A from Stockholm and two negative controls each
 +
            in a triplicate of which one is similar to the positive control but without 10 mM calcium in the reaction
 +
            buffer and one is without Sortase A in the reaction. </center>
 +
        </p>
 +
    </div>
 +
    <br><br>
 +
    <p>The gel in figure 3 shows a first proof for our suspicion. The first triplicate on the left contains CP-LPETGG,
 +
        GGGG-mCherry and Sortase from Stockholm in a buffer with 10 mM calcium present. The middle triplicate shows
 +
        CP-LPETGG, GGGG-mCherry and Sortase from Stockholm but in a buffer with no calcium present. The last triplicate
 +
        does only contain CP-LPETGG and GGGG-mCherry but no Sortase A from Stockholm. The reaction was performed at 37
 +
        °C for 90 minutes. As all three sample show the same outcome it can be assumend that the Sortase A from team
 +
        Stockholm does not show any activity whatsoever.<br>
 +
        In order to have a final proof we compared our Sortase A7M to the Sortase A from Stockholm using a FRET
 +
        connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP (fig. 4). We ran this
 +
        reaction for 3h at 30 °C.<br></p>
 +
    <img class="img-fluid center"
 +
        src="https://2019.igem.org/wiki/images/6/64/T--TU_Darmstadt--mCherry_GFP_FRET_different_sortases.png"
 +
        style=max-width:100%;>
 +
    <div class="caption">
 +
        <p>
 +
            <b>
 +
                <center> Figure 4:
 +
            </b>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 negeative control without Sortase. </center>
 +
        </p>
 +
    </div>
 +
 
 +
    <p>As visible in figure 4 the Sortase A from Stockholm does not show any activity during the reaction although it
 +
        was incubated with 10mM calcium present. In contrary to our Sortase A7M which was incubated without calcium
 +
        because it is an independent mutant. The Sortase A7M also 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 (data not shown). In comparison the Stockholm Sortase A 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-PAGEs that showed the same outcome of the Sortase A from Stockholm being not functional independent from the
 +
        presence of calcium.<br><br></p>
 +
 
 +
    <!-- -->
 +
    <span class='h3bb'>Sequence and Features</span>
 +
    <partinfo>BBa_K2144008 SequenceAndFeatures</partinfo>
  
  
−
<!-- Uncomment this to enable Functional Parameter display  
+
    <!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2144008 parameters</partinfo>
 
<partinfo>BBa_K2144008 parameters</partinfo>
 
<!-- -->
 
<!-- -->
  
−
===References===
+
    ===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
+
    [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
+
    [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

Revision as of 15:07, 18 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 FRET-based assay)

We purified the Sortase A from team Stockholm via fast protein liquid chromatography (FPLC) using the ÄKTA pure (fig. 2). In order to purify the Sortase A from team Stockholm we used the existing His-tag.

< ----- Bild von ÄKTA Chromatogram ---->

Fig 1: Chromatogram of the ÄKTA during the purification of Sortase A from team Stockholm. In figure 2 it is shown that the Sortase A from Stockholm eluted at ???. This should make sure that we have purified the right protein with the fitting tag.
We then confirmed the data of the purification by checking on the size of the Sortase using SDS-PAGE (fig. 2). We purified it twice with the same result in size.

   <img class="img-fluid center"
       src="20191018135731%21T--TU_Darmstadt--Klara_Sortase_Stockholm_Gr%C3%B6%C3%9Fe.png"
       style=max-width:80%;>

Figure 2: 
           </b>A SDS-PAGE 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. 
       </p>
   </div>

   <p>In figure 2 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 from Stockholm to reassure it has the right size of
       about 15 kDa which is the case. On the left are triplicates of coat protein with an LPETGG-tag (CP-LPETGG) and
       GGGG-mCherry. The first three are with Sortase Stockholm in the reaction and the middle three are without
       Sortase Stockholm. The reaction was run at 37 °C for 90 minutes. Seeing this first gel arose the suspicion that
       the Sortase A from team Stockholm is not working properly. We then tried to confirm whether our suspicion was
       right. In order to do so we ran another test with a SDS-PAGE (fig. 3) and additionally used the Sortase A from
       Stockholm for one of our self-made FRET reactions. 

 </p>
   <img class="img-fluid center"
       src="T--TU_Darmstadt--Klara_Sortase_Stockholm_Funktion.png"
       style=max-width:80%;>

       <p>
           <b>
Figure 3: 
           </b>A SDS-PAGE of the sortase reaction connecting CP-LPETGG and GGGG-mCherry. The gel includes a positive
           triplicate consisting of CP-LPETGG, GGGG-mCherry and Sortase A from Stockholm and two negative controls each
           in a triplicate of which one is similar to the positive control but without 10 mM calcium in the reaction
buffer and one is without Sortase A in the reaction. 
       </p>

   


   <p>The gel in figure 3 shows a first proof for our suspicion. The first triplicate on the left contains CP-LPETGG,
       GGGG-mCherry and Sortase from Stockholm in a buffer with 10 mM calcium present. The middle triplicate shows
       CP-LPETGG, GGGG-mCherry and Sortase from Stockholm but in a buffer with no calcium present. The last triplicate
       does only contain CP-LPETGG and GGGG-mCherry but no Sortase A from Stockholm. The reaction was performed at 37
       °C for 90 minutes. As all three sample show the same outcome it can be assumend that the Sortase A from team
       Stockholm does not show any activity whatsoever.

       In order to have a final proof we compared our Sortase A7M to the Sortase A from Stockholm using a FRET
       connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP (fig. 4). We ran this
       reaction for 3h at 30 °C.
</p>
   <img class="img-fluid center"
       src="T--TU_Darmstadt--mCherry_GFP_FRET_different_sortases.png"
       style=max-width:100%;>

       <p>
           <b>
Figure 4: 
           </b>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 negeative control without Sortase. 
       </p>

   <p>As visible in figure 4 the Sortase A from Stockholm does not show any activity during the reaction although it
       was incubated with 10mM calcium present. In contrary to our Sortase A7M which was incubated without calcium
       because it is an independent mutant. The Sortase A7M also 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 (data not shown). In comparison the Stockholm Sortase A 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-PAGEs that showed the same outcome of the Sortase A from Stockholm being not functional independent from the
       presence of calcium.

</p>

   Sequence and Features

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