Difference between revisions of "Part:BBa K2144008"

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	GGGG-mCherry. The first three are with Sortase Stockholm in the reaction and the middle three are without		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		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	+	the Sortase A from team Stockholm is not working properly. </p>
−	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>	+
−	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>	+
−	</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		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		connecting 5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) with GGGG-sfGFP (fig. 4). We ran this

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Revision as of 20:11, 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 assays)

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.

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.

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.

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.

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