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| | <h1>Results</h1> | | <h1>Results</h1> |
| | | | |
| − | <h2>FRET-based assay to analyze Sortase ligation efficiency</h2> | + | |
| − | <h1>Results</h1>
| + | |
| − | <h2>Characterization of Sortase A7M (and comparison to Sortase A5M)</h2>
| + | |
| − | <h3>How do we measure if our purified sortases are active?</h3>
| + | |
| − | <p>After purification of the sortases, we first performed SDS-PAGEs to verify that they are pure and
| + | |
| − | monomeric. You can see in <b>Fig. 3</b> that the purifications were successful. Next, we tested
| + | |
| − | if the purified sortases connect two proteins that carry the important Sortase-recognition tags,
| + | |
| − | N-terminal polyG and C-terminal LPETGG. Therefore, we added the sortases to a mix of
| + | |
| − | GGGG-mCherry and mCherry-LPETGG. The reactions were performed in different buffers, at different
| + | |
| − | enzyme-to-substrate ratios and for different time spans. We performed an SDS-PAGE, and prior to
| + | |
| − | Coomassie staining, we recorded fluorescent images of the gel. Thereby, we could identify
| + | |
| − | mCherry bands in the gel.
| + | |
| − | </p>
| + | |
| − | <img class="center"
| + | |
| − | src="https://2019.igem.org/wiki/images/5/59/T--TU_Darmstadt--SDS_Sortase_A7M_Sortase_A5M.png"
| + | |
| − | style="max-width:40%" />
| + | |
| − | </a>
| + | |
| − | <div class="caption">
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Figure x :
| + | |
| − | </b>
| + | |
| − | SDS-PAGE of Sortase A7M and Sortase A5M where the bands show up at
| + | |
| − | approximately 15 kDa. Our estimated size for Sortase A7M was 17.85 kDa,
| + | |
| − | and for Sortase A5M 18.07 kDa. This confirms the result shown on the gel,
| + | |
| − | since the band of Sortase A5M is a little higher than the one of Sortase A7M.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <img class="img-fluid center"
| + | |
| − | src="https://2019.igem.org/wiki/images/6/67/T--TU_Darmstadt--SDS_buff_fluor_Srt5_7_1%3B3_1%3B10_.png"
| + | |
| − | style="max-width:50%" />
| + | |
| − | </a>
| + | |
| − | | + | |
| − | <img class="img-fluid center"
| + | |
| − | src="https://2019.igem.org/wiki/images/0/0d/T--TU_Darmstadt--SDS_buff_both_Srt5_7_1%3B3_1%3B10_.png"
| + | |
| − | style="max-width:50%" />
| + | |
| − | </a>
| + | |
| − | <div class="caption">
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Figure x :
| + | |
| − | | + | |
| − | </b>
| + | |
| − | Fluorescence gel of the sortase-reaction of GGGG-mCherry and mCherry-LPETGG
| + | |
| − | mediated by Sortase A7M incubated for 2 h and
| + | |
| − | 4 h each. Reaction solutions were mixed with different ratios from enzyme to
| + | |
| − | substrate concentration(1:3;1:10) and each incubated in two different buffers(Tris-HCl
| + | |
| − | and Ammoniumdicarbonat).
| + | |
| − | Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from
| + | |
| − | left to right). The bands below the product at about 38 kDa could be semi-denatured
| + | |
| − | mCherry dimers.<br>
| + | |
| − | <b>b)</b> Fluorescence gel on top of the coomassie-stained gel of the sortase-reaction
| + | |
| − | of GGGG-mCherry and mCherry-LPETGG mediated by Sortase A7M incubated for 2 h
| + | |
| − | and
| + | |
| − | 4 h each. Reaction solutions were mixed with different ratios from enzyme to
| + | |
| − | substrate concentration(1:3;1:10) and each incubated in two different buffers (Tris-HCl
| + | |
| − | and Ammoniumdicarbonat).
| + | |
| − | Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from
| + | |
| − | left to right). The bands below the product at about 38 kDa could be semi-denatured
| + | |
| − | mCherry dimers. Additionally, Sortase A7M can be seen at 17 kDaA7M.The
| + | |
| − | unprocessed mCherry monomers can be seen at 28 kDa.
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | As shown in <b>Fig. 4</b>, under certain conditions, a product band appeared at the expected
| + | |
| − | size of 57.3 kDa (28.5+28.8 kDa). From this first activity test, we draw three
| + | |
| − | conclusions:
| + | |
| − | </p>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | | + | |
| − | <b>Our purified Sortase A7M is active</b>
| + | |
| − | | + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <b>The enzyme-substrate ratio affects the product yield</b>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <b>The duration of the reaction affects the product yield</b>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <p>
| + | |
| − | <br>
| + | |
| − | Additionally, TRIS buffer seems to alter the coomassie staining efficiency of Sortase A7M.
| + | |
| − | This endpoint measurement gave us a first impression that our Sortase A7M works nicely. Of
| + | |
| − | course, we wanted to further characterize the parameters of the reaction. When we understand
| + | |
| − | the Sortase better, modification of our VLPs will become more straightforward.
| + | |
| − | | + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <h3>how do we measure sortase reaction kinetics</h3>
| + | |
| − | <p>
| + | |
| − | In the above described assays, we noticed the impact of enzyme-substrate ratio and reaction
| + | |
| − | duration on the
| + | |
| − | overall product yield. We thought about how to further measure the kinetics of the sortase
| + | |
| − | reaction. In the
| + | |
| − | literature, sortase reaction kinetics are often measured by FRET-assays. Therefore, we designed
| + | |
| − | a suitable
| + | |
| − | FRET-assay. In the
| + | |
| − | end, we came
| + | |
| − | up with a new FRET pair not described in the literature to date: 5-TAMRA-LPETG and GGGG-sfGFP.
| + | |
| − | </p>
| + | |
| − | <h4>Development of a new FRET pair</h4>
| + | |
| − | <p>
| + | |
| − | For characterization of the reaction kinetics of Sortase A7M, Sortase A5M and
| + | |
| − | Sortase A, we
| + | |
| − | decided to develop a suitable FRET pair.
| + | |
| − | In order to find an optimal FRET pair, we first recorded an emission and absorption
| + | |
| − | spectrum of
| + | |
| − | 5-Carboxytetramethylrhodamin-LPETG (TAMRA) and GGGG-mCherry to verify the suitability for the
| + | |
| − | FRET effect, checking for a possible overlap between the donor's emission and the
| + | |
| − | acceptor's
| + | |
| − | extinction.
| + | |
| − | </p>
| + | |
| − | <img class="img-fluid center"
| + | |
| − | src="https://2019.igem.org/wiki/images/d/d4/T--TU_Darmstadt--TAMRA_mCherry.JPG"
| + | |
| − | style="max-width:70%" />
| + | |
| − | </a>
| + | |
| − | <div class="caption">
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Figure x :
| + | |
| − | </b>
| + | |
| − | Design of a FRET-pair of 5-TAMRA-LPETG (TAMRA) and GGGG-mCherry (mCherry). In
| + | |
| − | this configuration TAMRA acts as donor and mCherry as acceptor. When the two
| + | |
| − | fluorophores are not linked via the substrates of the sortase only TAMRA is being
| + | |
| − | excited. After sortase mediated ligation of the two substrates mCherry is the
| + | |
| − | fluorophore being excited via the FRET and the emission of mCherry intensifies.
| + | |
| − | Meanwhile, the emission of TAMRA decreases.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <p>
| + | |
| − | TAMRA is a chemical fluorophore that has an absorbance maximum at 542 nm and an emission
| + | |
| − | maximum at
| + | |
| − | 570 nm. The
| + | |
| − | terminal carboxy
| + | |
| − | group of the dye was linked via a lysine linker to the LPETG sequence (<b>see Fig. 5</b>).
| + | |
| − | mCherry has
| + | |
| − | an N-terminal poly-glycine sequence and can therefore be linked to the LPETG motif of TAMRA via
| + | |
| − | the
| + | |
| − | Sortase A. For a sufficient FRET-effect, it is also necessary that the distance between
| + | |
| − | donor and
| + | |
| − | acceptor is lower than the Förster radius. The Förster radius describes the distance between two
| + | |
| − | fluorophores at which 50 % of the energy is transferred.
| + | |
| − | <br>
| + | |
| − | First, we wanted to identify which concentrations are needed for our experiment, then set up the
| + | |
| − | reaction
| + | |
| − | and measured fluorescence intensities. Over time, a decline in the emission of TAMRA can be
| + | |
| − | observed as
| + | |
| − | Sortase A7M/A5M is converting more educts to products.
| + | |
| − | </p>
| + | |
| − | <img class="img-fluid center"
| + | |
| − | src="https://2019.igem.org/wiki/images/0/06/T--TU_Darmstadt--mCherry_TAMRA_Extinction_Emission.png"
| + | |
| − | style="max-width:50%" />
| + | |
| − | </a>
| + | |
| − | <div class="caption">
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Figure x :
| + | |
| − | </b>
| + | |
| − | The graph shows the extinction and emission spectra of TAMRA and mCherry. Due
| + | |
| − | to the large overlap of TAMRA emission and mCherry extinction it is possible to perform
| + | |
| − | a FRET with this pair of fluorophores. The graph show the relative fluorescence unit
| + | |
| − | (RFU[%]) in relation to the extincted/emitted wavelength [nm]. The peaks are normalized
| + | |
| − | to 100 %.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <p>
| + | |
| − | The emission and extinction spectra of TAMRA and mCherry exhibit an overlap of emission of TAMRA
| + | |
| − | and
| + | |
| − | extinction of mCherry. Based on this output, a FRET-assay for the kinetics of Sortase A7M
| + | |
| − | was performed
| + | |
| − | to confirm whether the FRET-pair is working.
| + | |
| − | As TAMRA is excited with light of a lower wavelength than mCherry, the former serves as FRET
| + | |
| − | donor and the
| + | |
| − | latter as acceptor. We chose the excitation wavelength at 485 nm to prevent unnecessary
| + | |
| − | “leak”
| + | |
| − | excitation of mCherry.
| + | |
| − | Nevertheless, an extinction of mCherry could not be excluded and may have negative effects on
| + | |
| − | the visibility
| + | |
| − | of the FRET.
| + | |
| − | </p>
| + | |
| − | <img class="img-fluid center"
| + | |
| − | src="https://2019.igem.org/wiki/images/b/b1/T--TU_Darmstadt--mCherry_TAMRA_Bleaching_Negativecontrol.png"
| + | |
| − | style="max-width:50%" />
| + | |
| − | </a>
| + | |
| − | | + | |
| − | <img class="img-fluid center"
| + | |
| − | src="https://2019.igem.org/wiki/images/f/fd/T--TU_Darmstadt--mCherry_TAMRA_Bleaching_Positive.png"
| + | |
| − | style="max-width:50%" />
| + | |
| − | </a>
| + | |
| − | <div class="caption">
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Figure x :
| + | |
| − | </b>
| + | |
| − | Spectrum of TAMRA and mCherry, with Sortase A7M, over the course of
| + | |
| − | 20 min in 5 min intervals. Depicted are the emission wavelengths against the
| + | |
| − | RFU. The sortase-mediated ligation results in a decline of both emission peaks.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <p>
| + | |
| − | The analysis of the data shown in <b>Fig. 7</b> confirmed the aforementioned
| + | |
| − | suspicion that mCherry is also excited at 485 nm, which makes differentiation
| + | |
| − | of the fluorescence more difficult. Furthermore, <b>Fig. 8</b> shows that the
| + | |
| − | difference in the decline of TAMRA is not significant. Accordingly, a decline in the
| + | |
| − | emission maximum of TAMRA over time is also visible in the negative control. One
| + | |
| − | reason might be bleaching of TAMRA through the excitation by the laser.
| + | |
| − | Nevertheless, conversion by the Sortase A7M can be observed by comparing the
| + | |
| − | results with the negative control.
| + | |
| − | </p>
| + | |
| − | <img class="img-fluid center"
| + | |
| − | src="https://2019.igem.org/wiki/images/9/92/T--TU_Darmstadt--mCherry_TAMRA_FRET_SortaseA7M.png"
| + | |
| − | style="max-width:60%" />
| + | |
| − | </a>
| + | |
| − | <div class="caption">
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Figure x :
| + | |
| − | </b>
| + | |
| − | Sortase reaction in TAMRA mCherry FRET after subtracting the negative control.
| + | |
| − | Depicted is the difference in RFU over time [min]. WIthin the first 20 min of the
| + | |
| − | substrate conversion is the quickest. At 30 min a plateau is reached. After
| + | |
| − | 60 min starts catalyzing the reverse reaction. The mean ΔRFU value was normalized
| + | |
| − | to zero for better visualization.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <h2>Comparison of MGGGG-mCherry and GGGG-mCherry (<a href="https://parts.igem.org/Part:BBa_K2868010"
| + | |
| − | target="_blank">BBa_K2868010</a></h2>
| + | |
| − |
| + | |
| − | | + | |
| − | | + | |
| − | </p>
| + | |
| − | <h2>References</h2>
| + | |
| − | <ol class="references">
| + | |
| − | <li id="cite_note-1">
| + | |
| − | <span class="mw-cite-backlink">
| + | |
| − | <a href="#cite_ref-1">↑</a>
| + | |
| − | </span>
| + | |
| − | <span class="reference-text">
| + | |
| − | Nathan Shaner, Robert Campbell, Paul Steinbach, Ben Giepmans, Amy Palmer and Roger Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology, 2004, 22: 1567-1572
| + | |
| − | <a rel="nofollow" class="external autonumber" href="#https://www.nature.com/articles/nbt1037">[1] </a>
| + | |
| − | </span>
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | </html> | | </html> |
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