Difference between revisions of "Part:BBa K3187019"

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	<h3>Usage and biology</h3>		<h3>Usage and biology</h3>
−	<p>Generally, the amino acid sequence LPXTG (X can be any amino acid) is a recognition sequence for the Sortase A.	+	<p>Generally, the amino acid sequence LPXTG (X can be any amino acid) is a recognition sequence for the Sortase&nbsp;A.
−	It is found in <i>S. aureus</i> and  is  important for anchoring exoproteins in the peptidoglycan layer.	+	It is found in <i>Staphylococcus&nbsp;aureus</i> and  is  important for anchoring exoproteins into the peptidoglycan layer.
−	Exoproteins are pathogenicity. LPXTG is recognized by sortase A a transpeptidase which cleaves between the threonine	+	Exoproteins are pathogenic. LPXTG is recognized by Sortase&nbsp;A, a transpeptidase which cleaves between the threonine
	and the glycine. The threonine forms an amid bond with the pentaglycine sequence of the peptidoglycan layer. As a result, a		and the glycine. The threonine forms an amid bond with the pentaglycine sequence of the peptidoglycan layer. As a result, a
−	covalently bond is produced. It  has been proven that the sequence LPETG is well recognized by sortase A	+	covalent bond is produced. It  has been proven, that the sequence LPETG is well recognized by Sortase&nbsp;A
	<sup id="cite_ref-1" class="reference">		<sup id="cite_ref-1" class="reference">
	<a href="#cite_note-1">[1] </a>		<a href="#cite_note-1">[1] </a>
	</sup>.		</sup>.
−	LPXTG is an easy opportunity to modify proteins, for example with peptides or other proteins that contains a polyG tag.	+	LPXTG is an easy opportunity to modify proteins, for example with peptides or other proteins, that contains a polyG&nbsp;tag.
	We used a tag with four glycines <a href="https://parts.igem.org/Part:BBa_K3187018"target="_blank">BBa_K3187018</a>.		We used a tag with four glycines <a href="https://parts.igem.org/Part:BBa_K3187018"target="_blank">BBa_K3187018</a>.
−	Thus VLPs are easily modified since they are made of proteins.	+	Thus, VLPs are easily modified since they are made of proteins.
−	<br>LPETGG is also possible recognized by sortase A. We used this sequence, since it was used in the publication,	+	<br>LPETGG can also be recognized by Sortase&nbsp;A. We used this sequence, as it was used in the publication
	we based our project on.		we based our project on.
	<sup id="cite_ref-2" class="reference">		<sup id="cite_ref-2" class="reference">
Line 43:		Line 43:
	</sup>		</sup>
	<br>LPETGG consists of six amino acids (lysine, proline, glutamine acid, threonine and two glycines)  The molecular weight		<br>LPETGG consists of six amino acids (lysine, proline, glutamine acid, threonine and two glycines)  The molecular weight
−	is 1.478 kDa.	+	is 1.478&nbsp;kDa.
	</p>		</p>
−	<p>LPETGG was linked to coat protein <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">BBa_K3187017</a> that	+	<p>LPETGG was linked to a coat protein <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">BBa_K3187017</a> to
	build the composite part coat protein with LPETGG tag (CP-LPETGG <a href="https://parts.igem.org/Part:BBa_K3187000"target="_blank">BBa_K3187000</a>)		build the composite part coat protein with LPETGG tag (CP-LPETGG <a href="https://parts.igem.org/Part:BBa_K3187000"target="_blank">BBa_K3187000</a>)
	and mCherry		and mCherry
−	<a href="https://parts.igem.org/Part:BBa_K3187026"target="_blank">BBa_K3187026</a>. Also mCherry with LPETGG tag is a composite part	+	<a href="https://parts.igem.org/Part:BBa_K3187026"target="_blank">BBa_K3187026</a>. mCherry with LPETGG tag also is a composite part
−	<a href="https://parts.igem.org/Part:BBa_K3187009"target="_blank">BBa_K3187009</a>	+	<a href="https://parts.igem.org/Part:BBa_K3187009"target="_blank">BBa_K3187009</a>.
	The LPETGG tag of coat protein was used to modify the coat protein with GGGG-mCherry and the assembeld Virus-like particles (VLPs).		The LPETGG tag of coat protein was used to modify the coat protein with GGGG-mCherry and the assembeld Virus-like particles (VLPs).
−	Furthermore, the tag enables us to control the functionality of Sortase A7M.	+	Furthermore, the tag enables us to control the functionality of Sortase&nbsp;A7M.
−	This was tested with the coupling of mCherry-LPETGG and GGGG-mCherry <a href="https://parts.igem.org/Part:BBa_K3187008"target="_blank">BBa_K3187008</a>.	+	This was tested by coupling mCherry-LPETGG and GGGG-mCherry <a href="https://parts.igem.org/Part:BBa_K3187008"target="_blank">BBa_K3187008</a> with each other.
	</p>		</p>
	<h3>Results</h3>		<h3>Results</h3>
−	<p>The SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry (Fig. 1) shows small red fluorescent bands when the concentration of sortase	+	<p>The SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry (<b>Fig.&nbsp;1</b>) shows small red fluorescent bands when the concentration of the Sortase&nbsp;A7M
−	is lower than 1:3. The bands are approximately found by 54 kDa. This suggests that two mCherrys are linked because the molecular	+	is lower than 1:3. The bands are approximately found at 54&nbsp;kDa. This suggests that two mCherrys are linked, as the molecular
−	weight is roughly doubled compared to the weight of one mCherry (27 kDa). Want to know more about the sortase ratio?	+	weight is roughly doubled, compared to the weight of one mCherry (27&nbsp;kDa). Want to know more about the sortase ratio?
−	Look at our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/Sortase"target="_blank">wiki</a>.	+	Have a look at our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/Sortase"target="_blank">wiki</a>.
	<div style="text-align: center;">		<div style="text-align: center;">
Line 69:		Line 69:
	<p>		<p>
	<b>Figure 1:</b>		<b>Figure 1:</b>
−	SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry with Sortase A7M.	+	SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry with Sortase&nbsp;A7M.
	</p>		</p>
	</div>		</div>
	</div>		</div>
	</p>		</p>
−	<p>Coat proteins with LPETGG assemble to Virus-like particles (VLPs). Images were made with the transmission electron microscopy (TEM)	+	<p>Coat proteins with LPETGG are able to assemble to Virus-like particles (VLPs). Images were taken with transmission electron microscopy (TEM),
−	and the diameter of VLPs was measured with dynamic light scattering.	+	and the diameter of the VLPs was measured with dynamic light scattering&nbsp;(DLS).
	<div style="text-align: center;">		<div style="text-align: center;">
	<a href="https://static.igem.org/mediawiki/parts/b/bc/T--TU_Darmstadt--TEM_CP_ohne_SP.jpeg"target="_blank">		<a href="https://static.igem.org/mediawiki/parts/b/bc/T--TU_Darmstadt--TEM_CP_ohne_SP.jpeg"target="_blank">
Line 96:		Line 96:
	<p>		<p>
	<b>Figure 5:</b>		<b>Figure 5:</b>
−	Diagram of DLS measurment of VLPs	+	Diagram of DLS measurment of VLPs.
	</p>		</p>
	</div>		</div>
	</div>		</div>
−	As shown in the diagram, VLPs which only consist of coat proteins with LPETGG tag and VLPs made out of coat proteins	+	As shown in the diagram, VLPs which only consist of coat proteins with LPETGG tag and VLPs made out of coat proteins
−	without a tag are smaller than P22-VLPs containing CP and SP (Fig. 5). Since, there is no difference between VLPs with and without LPETGG tag,	+	without a tag are smaller than P22-VLPs containing CP and SP (<b>Fig.&nbsp;5</b>). As we saw no difference in functionality between VLPs with and without LPETGG tag,
	the tag does not disturb the functionality of coat proteins.		the tag does not disturb the functionality of coat proteins.
	</p>		</p>
−	<p>For more information about CP-LPETGG, please look <a href="https://parts.igem.org/Part:BBa_K3187000"target="_blank">here</a>	+	<p>For more information about CP-LPETGG, please see <a href="https://parts.igem.org/Part:BBa_K3187000"target="_blank">here</a>.
	</p>		</p>
	</p>		</p>

---

Latest revision as of 16:58, 21 October 2019

LPETGG Tag for Sortase-mediated Ligation

Profile

Name	LPETGG
Base pairs	18
Molecular weight	1.478 kDa
Origin	Synthetic

Usage and biology

Generally, the amino acid sequence LPXTG (X can be any amino acid) is a recognition sequence for the Sortase A. It is found in Staphylococcus aureus and is important for anchoring exoproteins into the peptidoglycan layer. Exoproteins are pathogenic. LPXTG is recognized by Sortase A, a transpeptidase which cleaves between the threonine and the glycine. The threonine forms an amid bond with the pentaglycine sequence of the peptidoglycan layer. As a result, a covalent bond is produced. It has been proven, that the sequence LPETG is well recognized by Sortase A ^[1] . LPXTG is an easy opportunity to modify proteins, for example with peptides or other proteins, that contains a polyG tag. We used a tag with four glycines BBa_K3187018. Thus, VLPs are easily modified since they are made of proteins.
LPETGG can also be recognized by Sortase A. We used this sequence, as it was used in the publication we based our project on. ^[2]
LPETGG consists of six amino acids (lysine, proline, glutamine acid, threonine and two glycines) The molecular weight is 1.478 kDa.

LPETGG was linked to a coat protein BBa_K3187017 to build the composite part coat protein with LPETGG tag (CP-LPETGG BBa_K3187000) and mCherry BBa_K3187026. mCherry with LPETGG tag also is a composite part BBa_K3187009. The LPETGG tag of coat protein was used to modify the coat protein with GGGG-mCherry and the assembeld Virus-like particles (VLPs). Furthermore, the tag enables us to control the functionality of Sortase A7M. This was tested by coupling mCherry-LPETGG and GGGG-mCherry BBa_K3187008 with each other.

Results

The SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry (Fig. 1) shows small red fluorescent bands when the concentration of the Sortase A7M is lower than 1:3. The bands are approximately found at 54 kDa. This suggests that two mCherrys are linked, as the molecular weight is roughly doubled, compared to the weight of one mCherry (27 kDa). Want to know more about the sortase ratio? Have a look at our wiki.

Coat proteins with LPETGG are able to assemble to Virus-like particles (VLPs). Images were taken with transmission electron microscopy (TEM), and the diameter of the VLPs was measured with dynamic light scattering (DLS).

The hydrodynamic diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering (DLS) analysis.

As shown in the diagram, VLPs which only consist of coat proteins with LPETGG tag and VLPs made out of coat proteins without a tag are smaller than P22-VLPs containing CP and SP (Fig. 5). As we saw no difference in functionality between VLPs with and without LPETGG tag, the tag does not disturb the functionality of coat proteins.

For more information about CP-LPETGG, please see here.

References

1. ↑ Silvie Hansenová Maňásková , Kamran Nazmi, Alex van Belkum, Floris J. Bikker, Willem J. B. van Wamel, Enno C. I. Veerman, Synthetic LPETG-Containing Peptide Incorporation in the Staphylococcus aureus Cell-Wall in a Sortase A- and Growth Phase-Dependent Manner, plos one, 19.02.2014 [1]
2. ↑ Dustin Patterson,*,†Benjamin Schwarz,‡John Avera,‡Brian Western,†Matthew Hicks,†Paul Krugler,†Matthew Terra, †Masaki Uchida,‡Kimberly McCoy,‡and Trevor Douglas*,Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22Virus-like Particle, Bioconjugate Chemistry, 2017, 28, 2114−2124 [2]

Sequence and Features