Difference between revisions of "Part:BBa K3187000"

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P22 Bacteriophage Coat Protein with LPETGG Tag for Sortase-mediated Ligation

Profile

Name	Coat protein with LPETGG in pET24
Base pairs	1359
Molecular weight	49.0 kDa
Origin	Synthetic
Parts	Coat protein, LPETGG, T7 promoter, lac-operator, RBS, T7 terminator, Short Linker 5AA, Strep-tagII
Properties	Assembly with scaffold proteins to VLPs which can be modified exterior.

Usage and Biology

The P22 VLP originates from the temperate bacteriophage P22. Its natural host is Salmonella typhimurium. Since it was isolated half a century ago it has been characterized thoroughly and has become a paradigm system for temperate phages. To date, nearly everything is known about its lifecycle. Because of that and its specific properties it generates an accessible VLP platform. ^[1]

An assembled P22 VLP consists of 420 copies of coat protein (CP: BBa_K3187017) and 100 to 300 copies of scaffold protein (SP: BBa_K3187021). ^[2]
The shell of the VLP is formed by the 46.6 kDa CP. The coat protein occurs in one configuration, which contains a globular structure on the outer surface and an extended domain on the inner surface. Seven CPs arrange in asymmetric units, which form the icosahedral structure of the VLP.^[3]
The 18 kDa SP is required for an efficient assembly and naturally consists of 303 amino acids. It has been shown, that an N‑terminal truncated SP of 163 amino acids retains its assembly efficiency. The 3D‑structure is composed of segmented helical domains, with little or no globular core. In solution is a mixture of monomers and dimers present.^[4] When purified CPs and SPs are mixed, they self‑assemble into VLPs.

P22 VLPs occur as a procapsid after assembly. If the VLP is heated up to 60 °C, the CP rearranges, forming the expanded shell form (EX). This form has a diameter of about 58 nm and the volume is doubled compared to the one of the procapsid. The expanded shell form changes into the whiffleball form (WB) when heated further up to 70 °C. The whiffleball has 10 nm pores, while the procapsid or the expanded shell form only have 2 nm pores.^[5] Furthermore, the P22 VLP consists of SP and CP, but it also can assemble with only CPs. If it assembles without SP it can form two sizes of capsids. The small capsid is built as a T = 4 icosahedral lattice with a diameter between 195 Å and 240 Å. The larger capsid also has an icosahedral lattice, but it is formed as T = 7. T being the "triangulation number", a measure for capsid size and complexity. Moreover, it is like the wild type VLP, which includes the SP. The diameter of the wild type VLP, is between 260 Å and 306 Å. Each capsid consists of a 85 Å thick icosahedral shell made of CP.^[6]

The coat protein with LPETGG (CP-LPETGG BBa_K3187000) consists of 452 amino acids, which are encoded by 1359 DNA base pairs. The whole protein has a mass of 49.0 kDa. Its relevant parts are the coat protein (CP) (BBa_K3187017) and the LPETGG sequence (BBa_K3187019).
LPETGG is a synthetic sequence that is recognized by the enzyme family Sortase A and allows the coupling of CP with other peptides and proteins. For this, the sortase cleaves between the amino acids threonine (T) and glycine (G), and threonine forms an amide bond with another polyG sequence. ^[7] We used the Sortase A7M (BBa_K3187028) and Sortase A5M (BBa_K3187016). The used polyG recognition sequence is composed of four glycines (GGGG) (BBa_K3187018) ^[8]. The assembled VLPs which consits of CP-LPETGG can be modified with sortase.

Figure 1: Scheme of Sortase mediated P22-VLP modification.

Of course there are more parts necessary in order to express the CP‑LPETGG heterologously in E. coli BL21 (DE3). As a backbone, the pET24-backbone was used. The gene of the CP is transcribed into mRNA and then translated into an amino acid sequence, which arranges into the 3D structure of the protein. The T7 promoter (BBa_K3187029) is recognized by the T7 polymerase. In order to regulate the protein production, the lac‑operator (BBa_K3187029) was used. Furthermore, a RBS (BBa_K3187029) is in the construct and a Short Linker (5AA) (BBa_K3187030) is found between CP and LPETGG. The T7 terminator (BBa_K3187032) and Strep-tag II (BBa_K3187025) are located downstream of the coat protein CDS.

Methods

Cloning

The CP-LPETGG was cloned into the pET24-backbone with restriction and ligation . To do this, the CP‑LPETGG, as well as the T7 promoter and the lac‑operator sequence, was ordered from Integrated DNA Technologies (IDT). To verify the cloning, the sequence was controlled by sanger sequencing by Microsynth Seqlab.

Purification

The CP‑LPETGG was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. The used affinity tag was Strep-tag II.

SDS-PAGE and western blot

To verify that the CP-LPETGG was produced, a SDS-PAGE followed by a western blot was performed.

Forming multimers

To test whether coat proteins with a LPETGG tag form stable multimers, the concentration of CP‑LPETGG was increased in the presence of the protein BSA. The concentrated protein solution was heated up to 95 °C and a SDS-PAGE was performed to verify the stability of multimers.

Sortase-mediated Ligation

In order to characterize CP‑LPETGG, different assays were performed. The possibility of modifying the CP was tested with mCherry and Sortase A7M. The Sortase A7M successfully linked mCherry and CP‑LPETGG. The linkage was verified with a SDS‑PAGE. To identify whether the Sortase A7M or Sortase A5M produce multimers of coat proteins with LPETGG‑tag, CP‑LPETGG and Sortase A7M and Sortase A5M were incubated for 3 h at 37 °C. The development of multimeres was confirmed via SDS‑PAGE.

Assembly

The assembly was tested in vivo and in vitro. The assembled VLPs were collected with ultracentrifugation and were visualized with transmission electron microscopy (TEM). Therefore, the in vivo assembled VLPs are purified with size-exclusion chromatography (SEC) (Sephadex-100 column) ^[9] The diameter of VLPs was measured with dynamic light scattering (DLS) analysis.

Results

Cloning and Expression

The successful cloning was confirmed with sanger sequencing. The purification was documented with an chromatogram and the successful production of the VLPs was confirmed with a western blot.

Figure 2: Chromatogram of the purification of CP-LPETGG.

Figure 3: Enlargement of the chromatogram for purification of CP-LPETGG.

The chromatogram shows a peak for elution between 52 mL and 56 mL. The maximum is found approximatley at 380 mAU (Fig. 2 and 2)).

Figure 4: Western blot of all produced and purified proteins.

Fig. 4 shows that the band of the CP‑LPETGG is can be seen at approximately 49 kDa. Consequently, the successful production was proven. CP‑LPETGG was detected with Strep‑Tactin‑HRP.

Forming multimers

The SDS‑PAGE (Fig. 5) suggests that the CP-LPETGG does not form multimers , even if it is in high concentration.

Figure 5: SDS-PAGE of BSA and CP‑LPETGG.

Fig. 5 shows the bands of BSA at approximately 66 kDa and the CP-LPETGG at approximately 49 kDa. When the proteins were combined, two bands can be seen, one of CP‑LPETGG and one of BSA. Hence, no multimers were formed.

Sortase-medited Reaction

The possibility of modification was shown with a SDS-PAGE, which shows GGGG‑mCherries linked to several CP‑LPETGG.

Figure 6: SDS-PAGE of CP-LPETGG modified with mCherry by Sortase A7M and Sortase A5M.

The SDS-PAGE shows multiple bands (Fig. 6), which relate to a higher molecular weight than mCherry or CP‑LPETGG themselves have. The bands located between 55 kDa and 70 kDa most likley show the linked CP‑LPETGG and GGGG‑mCherry as we expected the product to be approximately .... kDa.

The results on the SDS-PAGE of testing sortase-mediated linkage between coat proteins with LPETGG-tag with Sortase A7M and Sortase A5M (Fig. 2) suggested that Sortase A7M and Sortase A5M produce CP-LPETGG multimers, because wild type Sortase A is able to link two proteins together via disulfide bridges ^[10] ^[11] and the P22 Coat Protein accommodates a cysteine residue (Fig. 7).

Figure 7: SDS-PAGE of sortase-mediated linkage between several coat proteins with LPETGG-tag.

Want to know more about sortase-mediated ligation? Please have a look at our wiki.

Assembly

Ultracentrifugation was used to harvest VLPs after in vivo and in vitro assembly

In vivo assembled VLPs

For extracting the VLPs, which consits of SP and with sfGFP modified CP‑LPETGG, directly from cell broth we first lysed the cells by sonication and got rid of debris by two centrifugation steps at 12,000 x g. Afterwards ultracentrifugation with a sucrose cushion (35% w/v) at 150,000 x g was used as a first concentration step. The resulting sediment contained fluorescent material which we suspected to contain a concentrated fraction of VLPs.

Figure 8: Cell broth after ultracentrifugation. Supernatant containing sfGFP‑SP and CP while VLPs collected in the sediment

Ultracentrifugation sediment most likely still contains monomeric proteins and small amounts of cell debris that can be harmful in some applications due to high endotoxin levels. For getting rid of these contaminants we subsequently used size-exclusion chromatography (SEC) (Sephadex‑100 column). ^[12] After SEC the elution sample with the highest suspected VLP concentration (based on UV absorption) was imaged with transmission electron microscopy (TEM). Numerous capsids in the correct size range were clearly visible. This lead us to believe that ultracentrifugation, as well as SEC treatment, do not interfere with capsid integrity while separating VLPs from other contaminants. Chromatography dilutes the sample significantly which is not optimal for analytic purposes. This is why a second ultracentrifugation treatment would be required for re-concentration of purified capsids as ^[12] suggest.

Figure 9: Intact P22-VLPs after size exclusion chromatography

in vitro assembled VLPs

The images of ultracentrifugation show that monomeric proteins were separated from assembled capsids by ultracentrifugation at 150.000 x g in a sucrose cushion (35% w/v). After completion of the ultracentrifugation treatment, a sediment was clearly visible in the centrifuge tube, which we suspected to mainly contain VLPs. TEM was used to image capsids taken from the sediment. For increased contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually intact VLPs all over the sample, measuring a diameter of 60 nm or less (Fig. 2).

Figure 10: Ultracentrifugation of in vitroassembled VLPs

The images taken via TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. Therefore, the CPs produced using this part must be fully functional. The CPs assemble with SPs and can be modified on the surface (Fig. 10). Moreover, CPs also assemble without SPs (Fig. = 11).

Figure 11: Assembly of only coat proteins with a LPETGG-tag.

Fig. 11 shows that no scaffold proteins are necessary for assembly.

Figure 12: Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.

Fig. 12 shows that CP-LPETGG and SPs assemble to VLPs and that CP-LPETGG can be modified for this process.

For more information about VLP assembly, please visit our wiki.

The hydrodynamic diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering (DLS) analysis. In general hydrodynamic diameters depend on several properties like polarity and charges as well as size and shape. These properties can be summed up as the electrical properties of the system. ^[13].

Figure 13: Influence of particle charge on hydrodynamic diameter.

Figure 14: Diagram of DLS measurment of VLPs .

We showed by dynamic light scattering (DLS) analysis (Fig. 5) that capsids containing only CP are smaller than P22-VLPs containing both CP and SP. This was also confirmed by measuring VLPs and CP-only capsids in TEM images using ImageJ. Capsids which are only composed of CP measured average diameter of 53 nm±4.3 nm are significantly smaller than VLPs out of SP and CP measured average diameter of 57 nm±3 nm (n=20; p < 0.005). What also became clear is that the presence of the LPETGG tag does not affect the size of the assembled CP-only capsid.

When we started to compare sfGFP-modified VLPs with non-modified VLPs using dynamic light scattering (DLS), we expected a difference in hydrodynamic radii because surface modifications should further increase the hydration of the particles as shown in Fig. 3^[14].

Figure 15: Dynamic light scattering analysis. Hydrodynamic diameters of different P22-VLP species.

As described here, non-modified VLPs showed hydrodynamic diameters of approximately 112.4 nm ± 41.3 nm. In comparison, modified capsids showed an average hydrodynamic diameter of 1446 nm. In our case, the drastically elevated hydrodynamic diameter of the P22-VLP linked to sfGFP may result from strong hydration since wild type sfGFP is multiply negatively charged ^[15]. This probably leads to a tremendous charge density all over the surface. Another possible reason could be the formation of sfGFP dimers attached to the VLPs.

In order to demonstrate the integrity of our modified VLPs we used capsids from the same sample for DLS and electron microscopy which confirms the presence of intact VLPs. The size distribution shows that they still pose a monodisperse species, even though their hydrodynamic diameter is increased compared to unmodified VLPs or capsids containing only CP.

For more information about VLP assembly, please visit our wiki.

References

↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [1]
↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412‑10414 [2]
↑ Wen Jiang, Zongli Li, Zhixian Zhang, Matthew Baker, Peter Prevelige Jr., and Wah Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nature Structural Biology, 2003, 10: 131‑135 [3]
↑ Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein, Journal of Molecular Biology, 1998, Volume 281: 69‑79 [4]
↑ Dustin Patterson, Peter Prevelige, Trevor Douglas, Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22, American Chemical Society, 2012, 6: 5000-5009 [5]
↑ P A Thuman-Commike, B Greene, J A Malinski, J King, and W Chiu, Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures., Biophysical Journal, 1998, 74: 559-568 [6]
↑ 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 [7]
↑ Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [8]
↑ Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [9]
↑ Jia X, Kwon S, Wang CI, Huang YH, Chan LY, Tan CC, Rosengren KJ, Mulvenna JP, Schroeder CI, Craik DJ, Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A, Journal of biological chemistry, 2014, 289, 627-6638 [10]
↑ Melissa E. Reardon-Robinson, Jerzy Osipiuk, Chungyu Chang, Chenggang Wu, Neda Jooya, Andrzej Joachimiak, Asis Das, Hung Ton-That‡2, A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris, Journal of biological chemistry, 2015, 290, 21393-21405 [11]
↑ Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [12]
↑ J. Rybka, A. Mieloch, A. Plis, M. Pyrski, T. Pnioewski and M.Giersig, Assambly and Characterization ofHBc Derived Virsus-like Particles with Magnetic Core, Nanomaterials (Basel), 2019, 9(2): 155 [13]
↑ https://www.horiba.com/uk/ scientific/ products/ particle-characterization/ applications/ pharmaceuticals/ viruses-virus-like-particles/ [14]
↑ Laber, J. R., Dear, B. J., Martins, M. L., Jackson, D. E., DiVenere, A., Gollihar, J. D., ... & Maynard, J. A. (2017). Charge shielding prevents aggregation of supercharged GFP variants at high protein concentration. Molecular pharmaceutics, 14(10), 3269-3280. [15]

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1491
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

[1] Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, pp 80- 88 [1]

[2] Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412‑10414 [2]

[3] Wen Jiang, Zongli Li, Zhixian Zhang, Matthew Baker, Peter Prevelige Jr., and Wah Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nature Structural Biology, 2003, 10: 131‑135 [3]

[4] Matthew Parker, Sherwood Casjens, Peter Prevelige Jr., Functional domains of bacteriophage P22 scaffolding protein, Journal of Molecular Biology, 1998, Volume 281: 69‑79 [4]

[5] Dustin Patterson, Peter Prevelige, Trevor Douglas, Nanoreactors by Programmed Enzyme Encapsulation Inside the Capsid of the Bacteriophage P22, American Chemical Society, 2012, 6: 5000-5009 [5]

[6] P A Thuman-Commike, B Greene, J A Malinski, J King, and W Chiu, Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures., Biophysical Journal, 1998, 74: 559-568 [6]

[7] 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 [7]

[8] Dustin Patterson, Benjamin LaFrance, Trevor Douglas, Rescuing recombinant proteins by sequestration into the P22 VLP, Chemical Communications, 2013, 49: 10412-10414 [8]

[9] Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [9]

[10] Jia X, Kwon S, Wang CI, Huang YH, Chan LY, Tan CC, Rosengren KJ, Mulvenna JP, Schroeder CI, Craik DJ, Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A, Journal of biological chemistry, 2014, 289, 627-6638 [10]

[11] Melissa E. Reardon-Robinson, Jerzy Osipiuk, Chungyu Chang, Chenggang Wu, Neda Jooya, Andrzej Joachimiak, Asis Das, Hung Ton-That‡2, A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris, Journal of biological chemistry, 2015, 290, 21393-21405 [11]

[12] Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009. [12]

[13] J. Rybka, A. Mieloch, A. Plis, M. Pyrski, T. Pnioewski and M.Giersig, Assambly and Characterization ofHBc Derived Virsus-like Particles with Magnetic Core, Nanomaterials (Basel), 2019, 9(2): 155 [13]

[14] ttps://www.horiba.com/uk/ scientific/ products/ particle-characterization/ applications/ pharmaceuticals/ viruses-virus-like-particles/ [14]

[15] Laber, J. R., Dear, B. J., Martins, M. L., Jackson, D. E., DiVenere, A., Gollihar, J. D., ... & Maynard, J. A. (2017). Charge shielding prevents aggregation of supercharged GFP variants at high protein concentration. Molecular pharmaceutics, 14(10), 3269-3280. [15]

[1]

[2]

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@@ Line 10: / Line 10: @@
                  <h3>Profile</h3>
-                 <table style=“width:80%“>
+                 <table style="width:80%">
                  <tr>
                  <td><b>Name</b></td>
@@ Line 39: / Line 39: @@
                  <h3> Usage and Biology</h3>
-                 <p>The P22&nbsp;VLP originates from the temperate bacteriophage&nbsp;P22. Its natural host is <i>Salmonella  typhimurium</i>.
+                 <p>The P22 VLP originates from the temperate bacteriophage P22. Its natural host is <i>Salmonella&nbsp;typhimurium</i>.
-                     Since it
+                     Since it was isolated half a century ago it has been characterized thoroughly and has become a paradigm system for temperate phages.
-                        was isolated half a century ago it has been characterized thoroughly and has become a paradigm system for
+                    To date, nearly everything is known about its lifecycle. Because of that and its specific properties it generates
-                        temperate phages. To date, nearly everything is known about its lifecycle including virions. Because of that
+                    an accessible VLP platform.
-                        and its specific properties it generates an accessible VLP platform.
+                    <sup id="cite_ref-1" class="reference">
-                        <sup id="cite_ref-1" class="reference">
+                                 <a href="#cite_note-1">[1]</a></sup><br>
-                                 <a href="#cite_note-1">[1]
+                    </p>
-                                 </a>
+                    <p>An assembled P22 VLP consists of 420&nbsp;copies of coat protein (CP: <a href="https://parts.igem.org/Part:BBa_K3187017" target="_blank">BBa_K3187017</a>) and 100 to 300 copies of scaffold
-                        </sup>
+                    protein (SP: <a href="https://parts.igem.org/Part:BBa_K3187021" target="_blank">BBa_K3187021</a>).
+                    <sup id="cite_ref-2" class="reference">
-                        <br>An assembled P22 VLP consists of 420 copies of coat protein (CP <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">BBa_K3187017</a>)
+                                 <a href="#cite_note-2">[2]</a>
-                        and 100 to 300 copies of scaffold protein (SP <a href="https://parts.igem.org/Part:BBa_K3187021"target="_blank">BBa_K3187021</a>).
+                            </sup><br>
-                        <sup id="cite_ref-2" class="reference">
+                    The shell of the VLP is formed by the 46.6&nbsp;kDa&nbsp;CP. The  coat protein occurs in one configuration, which contains a globular
-                                 <a href="#cite_note-2">[2]
+                    structure on the outer surface and an extended domain on the inner surface. Seven CPs arrange in asymmetric units, which form
-                                 </a>
+                    the icosahedral structure of the VLP.<sup id="cite_ref-3" class="reference">
-                        </sup>
+                                 <a href="#cite_note-3">[3]</a>
-                        The shell of the VLP is formed by the 46.6&nbsp;kDa CP. The gene 5 coat protein occurs in one configuration, which contains
+                            </sup><br>
-                        a globular structure on the outer surface and an extended domain on the inner surface. Seven CPs arrange in asymmetric
+                    The 18&nbsp;kDa&nbsp;SP is required for an efficient assembly and naturally consists of 303&nbsp;amino acids. It has been shown, that an
-                        units, which form the icosahedral structure of the VLP.
+                    N&#8209;terminal truncated SP of 163 amino acids retains its assembly efficiency. The 3D&#8209;structure is composed of segmented helical
-                        <sup id="cite_ref-3" class="reference">
+                    domains, with little or no globular core. In solution is a mixture of monomers and dimers present.<sup id="cite_ref-4"
-                                 <a href="#cite_note-3">[3]
+                                class="reference">
-                                 </a>
+                                 <a href="#cite_note-4">[4]</a>
-                        </sup>
+                            </sup>
-                        The 18&nbsp;kDa SP is required for an efficient assembly and naturally consists of 303&nbsp;amino&nbsp;acids. It has been shown, that an
+                    When purified CPs and SPs are mixed, they self&#8209;assemble into VLPs. </p>
-                        N-terminal truncated SP of 163 amino acids retains its assembly efficiency. The 3D-structure is composed of segmented helical
-                        domains, with little or no globular core. In solution is a mixture of monomers and dimers present.
+                    <p> P22 VLPs occur as a procapsid after assembly. If the VLP is heated up to 60&nbsp;°C, the CP rearranges, forming
-                        <sup id="cite_ref-4" class="reference">
+                    the expanded shell form&nbsp;(EX). This form has a diameter of about 58&nbsp;nm and the volume is doubled compared to the one of
-                                 <a href="#cite_note-4">[4]
+                    the procapsid. The expanded shell form changes into the whiffleball form (WB) when heated further up to 70 &nbsp;°C. The
-                                 </a>
+                    whiffleball has 10&nbsp;nm pores, while the procapsid or the expanded shell form only have 2&nbsp;nm pores.<sup id="cite_ref-5" class="reference">
-                        </sup>
+                            <a href="#cite_note-5">[5]</a>
-                        If purified CPs and SPs are mixed, they assemble into VLPs.
-                        <br>P22 VLPs occur as a procapsid after assembly. If the VLP is heated up to 60&nbsp;degrees, the CP rearranges, forming
-                        the expanded shell form (EX). This form has a diameter of about 58 nm and the volume is doubled compared to the one of
-                        the procapsid. The expanded shell form changes into the wiffleball form (WB) when heated further up to 70 degrees. The
-                        wiffleball has 10&nbsp;nm pores, while the procapsid or the expanded shell form only have 2&nbsp;nm pores.
-                        <sup id="cite_ref-5" class="reference">
-                                <a href="#cite_note-5">[5]
-                                 </a>
-                        </sup>
-                         Furthermore, the
-                        P22 VLP consists of SP and CP, but it also can assemble only with CPs. If it assembles without SP it can form two sizes of
-                        capsids. The small capsid is built as a T&nbsp;=&nbsp;4 icosahedral lattice with a diameter between 195&nbsp;Å and 240&nbsp;Å. The larger capsid
-                        also has an icosahedral lattice, but it is formed as T&nbsp;=&nbsp;7. Moreover, it is like the wild type VLP, which includes the SP. The
-                        diameter of the wild type VLP is between 260&nbsp;Å and 306&nbsp;Å. Each capsid consists of a 85&nbsp;Å thick icosahedral
-                        shell out of CP.
-                        <sup id="cite_ref-6" class="reference">
-                                <a href="#cite_note-6">[6]
-                                 </a>
                          </sup>
+                    Furthermore, the P22 VLP consists of SP and CP, but it also can assemble with only CPs. If it assembles without SP it can form
+                    two sizes of capsids. The small capsid is built as a T&nbsp;=&nbsp;4 icosahedral lattice with a diameter between 195&nbsp;Å and 240&nbsp;Å. The
+                    larger capsid also has an icosahedral lattice, but it is formed as T&nbsp;=&nbsp;7. T being the "triangulation number", a measure for
+                    capsid size and complexity. Moreover, it is like the wild type VLP, which includes the SP. The diameter of the wild type VLP, is
+                    between 260&nbsp;Å and 306&nbsp;Å. Each capsid consists of a 85&nbsp;Å thick icosahedral shell made of CP.<sup id="cite_ref-6" class="reference">
+                            <a href="#cite_note-6">[6] </a>
+                        </sup>
                      </p>
-                 <p>The coat protein with LPETGG (CP-LPETGG <a href="https://parts.igem.org/Part:BBa_K3187000"target="_blank">BBa_K3187000)</a>
+                 <p>The coat protein with LPETGG (CP-LPETGG <a href="https://parts.igem.org/Part:BBa_K3187000" target="_blank">BBa_K3187000)</a>
                      consists of 452&nbsp;amino&nbsp;acids, which are encoded by 1359&nbsp;DNA&nbsp;base&nbsp;pairs. The whole
-                     protein has a mass of 49.0&nbsp;kDa. Its relevant parts are the coat protein (CP) <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">(BBa_K3187017)</a>
+                     protein has a mass of 49.0&nbsp;kDa. Its relevant parts are the coat protein (CP) <a href="https://parts.igem.org/Part:BBa_K3187017" target="_blank">(BBa_K3187017)</a>
-                     and the LPETGG sequence <a href="https://parts.igem.org/Part:BBa_K3187019"target="_blank">(BBa_K3187019)</a>.
+                     and the LPETGG sequence <a href="https://parts.igem.org/Part:BBa_K3187019" target="_blank">(BBa_K3187019)</a>.
                      <br>LPETGG is a synthetic sequence that is recognized by the enzyme family Sortase&nbsp;A
                      and allows the coupling of CP with other peptides and proteins. For this, the sortase
@@ Line 103: / Line 91: @@
                               </a>
                      </sup>
-                     We used the Sortase&nbsp;A7M <a href="https://parts.igem.org/Part:BBa_K3187028"target="_blank">(BBa_K3187028)</a>
+                     We used the Sortase&nbsp;A7M <a href="https://parts.igem.org/Part:BBa_K3187028" target="_blank">(BBa_K3187028)</a>
-                     and Sortase&nbsp;A5M <a href="https://parts.igem.org/Part:BBa_K3187016"target="_blank">(BBa_K3187016)</a>.
+                     and Sortase&nbsp;A5M <a href="https://parts.igem.org/Part:BBa_K3187016" target="_blank">(BBa_K3187016)</a>.
-                     The used polyG recognition sequence is composed of four glycines (GGGG) <a href="https://parts.igem.org/Part:BBa_K3187018"target="_blank">(BBa_K3187018)</a>
+                     The used polyG recognition sequence is composed of four glycines (GGGG) <a href="https://parts.igem.org/Part:BBa_K3187018" target="_blank">(BBa_K3187018)</a>
@@ Line 111: / Line 99: @@
                         <a href="#cite_note-8">[8]
                          </a>
-                     </sup>
+                     </sup>. The assembled VLPs which consits of CP-LPETGG can be modified with sortase.
+                </p>
+                <a href="https://2019.igem.org/wiki/images/a/aa/T--TU_Darmstadt--modification_with_sfGFP.png"
+                target="_blank"
+              >
+                <img
+                  class="img-fluid center"
+                  src="https://2019.igem.org/wiki/images/a/aa/T--TU_Darmstadt--modification_with_sfGFP.png"
+                  style="max-width:80%;"
+                />
+              </a>
+              <div class="caption">
+                <p>
+                  <b> Figure 1:</b>
+                  Scheme of Sortase mediated P22-VLP modification.
                  </p>
+              </div>
+            </div>
                  <p>Of course there are more parts necessary in order to express the CP&#8209;LPETGG heterologously in
                      <i>E.&nbsp;coli</i> BL21 (DE3). As a backbone, the pET24-backbone was used. The gene of the CP is transcribed
                      into mRNA and then translated into an amino acid sequence, which arranges into the 3D structure of the protein.
-                     The T7&nbsp;promoter <a href="https://parts.igem.org/Part:BBa_K3187029"target="_blank">(BBa_K3187029)</a>
+                     The T7&nbsp;promoter <a href="https://parts.igem.org/Part:BBa_K3187029" target="_blank">(BBa_K3187029)</a>
                      is recognized by the T7&nbsp;polymerase. In order to regulate the protein production, the
-                     <i>lac</i>-operator <a href="https://parts.igem.org/Part:BBa_K3187029"target="_blank">(BBa_K3187029)</a> was used.
+                     <i>lac</i>&#8209;operator <a href="https://parts.igem.org/Part:BBa_K3187029" target="_blank">(BBa_K3187029)</a> was used.
-                     Furthermore, a RBS <a href="https://parts.igem.org/Part:BBa_K3187029"target="_blank">(BBa_K3187029)</a> is in the construct and
+                     Furthermore, a RBS <a href="https://parts.igem.org/Part:BBa_K3187029" target="_blank">(BBa_K3187029)</a> is in the construct and
-                     a Short&nbsp;Linker&nbsp;(5AA) <a href="https://parts.igem.org/Part:BBa_K3187030"target="_blank">(BBa_K3187030)</a>
+                     a Short&nbsp;Linker&nbsp;(5AA) <a href="https://parts.igem.org/Part:BBa_K3187030" target="_blank">(BBa_K3187030)</a>
-                     is found between CP and LPETGG. The T7&nbsp;terminator <a href="https://parts.igem.org/Part:BBa_K3187032"target="_blank">(BBa_K3187032)</a> and
+                     is found between CP and LPETGG. The T7&nbsp;terminator <a href="https://parts.igem.org/Part:BBa_K3187032" target="_blank">(BBa_K3187032)</a> and
-                     Strep-tag&nbsp;II <a href="https://parts.igem.org/Part:BBa_K3187025"target="_blank">(BBa_K3187025)</a> are
+                     Strep-tag&nbsp;II <a href="https://parts.igem.org/Part:BBa_K3187025" target="_blank">(BBa_K3187025)</a> are
                      located downstream of the coat&nbsp;protein&nbsp;CDS.
                  </p>
@@ Line 128: / Line 136: @@
                  <h3> Methods</h3>
                  <h4>Cloning</h4>
-                 <p>The CP-LPETGG was cloned into the pET24-backbone with <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">restriction and ligation</a> .
+                 <p>The CP-LPETGG was cloned into the pET24-backbone with <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">restriction and ligation</a> .
                      To do this, the CP&#8209;LPETGG, as well as the T7&nbsp;promoter and the
-                     <i>lac</i>-operator sequence, was ordered from Integrated&nbsp;DNA&nbsp;Technologies&nbsp;(IDT). To verify the cloning,
+                     <i>lac</i>&#8209;operator sequence, was ordered from Integrated&nbsp;DNA&nbsp;Technologies&nbsp;(IDT). To verify the cloning,
                      the sequence was controlled by sanger&nbsp;sequencing by Microsynth&nbsp;Seqlab.
                  </p>
                  <h4>Purification</h4>
-                 <p>The CP-LPETGG was heterologously expressed in <i>E.&nbsp;coli</i> BL21 and purified with
+                 <p>The CP&#8209;LPETGG was heterologously expressed in <i>E.&nbsp;coli</i> BL21 and purified with
-                      <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">GE&nbsp;Healthcare&nbsp;ÄKTA&nbsp;Pure&nbsp;machine</a>
+                      <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">GE&nbsp;Healthcare&nbsp;ÄKTA&nbsp;Pure&nbsp;machine</a>
                       which is a machine for FPLC. The used affinity tag was Strep-tag&nbsp;II.
                  </p>
                  <h4>SDS-PAGE and western blot</h4>
-                 <p>To verify that the CP-LPETGG was produced, a  <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">SDS-PAGE</a> followed by a
+                 <p>To verify that the CP-LPETGG was produced, a  <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">SDS-PAGE</a> followed by a
-                     <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">western&nbsp;blot</a> was performed.
+                     <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">western&nbsp;blot</a> was performed.
                  </p>
                  <h4>Forming multimers</h4>
@@ Line 149: / Line 157: @@
                  <h4>Sortase-mediated Ligation</h4>
                   <p>In order to characterize CP&#8209;LPETGG, different assays were performed. The possibility of modifying the CP was tested with
-                     mCherry and Sortase&nbsp;A7M. The Sortase&nbsp;A7M successfully linked mCherry and CP-LPETGG.
+                     mCherry and Sortase&nbsp;A7M. The Sortase&nbsp;A7M successfully linked mCherry and CP&#8209;LPETGG.
                      The linkage was verified with a SDS&#8209;PAGE.
-                     To identify whether the Sortase A7M or Sortase A5M
+                     To identify whether the Sortase&nbsp;A7M or Sortase&nbsp;A5M
 <!-- WURDEN HIER BEIDE SORTASEN UNTERSUCHT?? -->
@@ Line 157: / Line 165: @@
-produce multimers of coat&nbsp;proteins with LPETGG-tag, CP-LPETGG and Sortase&nbsp;A7M and Sortase&nbsp;A5M
+produce multimers of coat&nbsp;proteins with LPETGG&#8209;tag, CP&#8209;LPETGG and Sortase&nbsp;A7M and Sortase&nbsp;A5M
@@ Line 168: / Line 176: @@
                  <h4>Assembly</h4>
                  <p> The assembly was tested <i>in&nbsp;vivo</i> and <i>in&nbsp;vitro</i>. The assembled VLPs were collected with
-                       <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">ultracentrifugation</a> and
+                       <a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">ultracentrifugation</a> and
-                       were visualized with transmission&nbsp;electron&nbsp;microscopy&nbsp;<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">(TEM)</a>.
+                       were visualized with transmission&nbsp;electron&nbsp;microscopy&nbsp;<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">(TEM)</a>.
                      Therefore, the <i>in vivo</i> assembled VLPs are purified with size-exclusion chromatography
-                     (<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf"target="_blank">SEC</a>)
+                     (<a href="https://static.igem.org/mediawiki/2019/6/62/T--TU_Darmstadt--Methoden.pdf" target="_blank">SEC</a>)
                      (Sephadex-100 column)
-                     <sup id="cite_ref-11" class="reference">
+                     <sup id="cite_ref-9" class="reference">
-                             <a href="#cite_note-11">[11]
+                             <a href="#cite_note-9">[9]
                               </a>
                           </sup>
@@ Line 184: / Line 192: @@
                  <h4>Cloning and Expression</h4>
-                 <p>The successful cloning was confirmed with sanger&nbsp;sequencing and successful production of the VLPs was confirmed with a western&nbsp;blot.
+                 <p>The successful cloning was confirmed with sanger&nbsp;sequencing. The purification was documented with an chromatogram and the
+                    successful production of the VLPs was confirmed with a western&nbsp;blot.
+                </p>
+                <div style="text-align: center;">
+                        <a href="https://2019.igem.org/wiki/images/f/f0/T--TU_Darmstadt--Chrom_CP-LPETGG.png" target="_blank">
+                      <img class="img-fluid center" src="https://2019.igem.org/wiki/images/f/f0/T--TU_Darmstadt--Chrom_CP-LPETGG.png" style="max-width:60%" />
+                      </a>
+                      <div class="caption">
+                         <p>
+                         <b>Figure 2:</b>
+                              Chromatogram of the purification of CP-LPETGG.
+                          </p>
+                      </div>
+                       </div>
+                <div style="text-align: center;">
+                        <a href="https://2019.igem.org/wiki/images/0/03/T--TU_Darmstadt--Chrom_CP-LPETGG-Peak.png" target="_blank">
+                      <img class="img-fluid center" src="https://2019.igem.org/wiki/images/0/03/T--TU_Darmstadt--Chrom_CP-LPETGG-Peak.png" style="max-width:60%" />
+                      </a>
+                      <div class="caption">
+                         <p>
+                         <b>Figure 3:</b>
+                              Enlargement of the chromatogram for purification of CP-LPETGG.
+                          </p>
+                      </div>
+                       </div>
+                       <p>The chromatogram shows a peak for elution between 52&nbsp;mL and 56&nbsp;mL. The maximum
+                    is found approximatley at 380 mAU (<b>Fig. 2 and 2)</b>).</p>
                          <div style="text-align: center;">
-                                 <a href="https://2019.igem.org/wiki/images/9/9c/T--TU_Darmstadt--Western_blot_CP-LPETGG_CP.jpeg"target="_blank">
+                                 <a href="https://2019.igem.org/wiki/images/9/9c/T--TU_Darmstadt--Western_blot_CP-LPETGG_CP.jpeg" target="_blank">
                                <img class="img-fluid center" src="https://2019.igem.org/wiki/images/9/9c/T--TU_Darmstadt--Western_blot_CP-LPETGG_CP.jpeg" style="max-width:60%" />
                                </a>
@@ Line 193: / Line 233: @@
                                <div class="caption">
                                   <p>
-                                  <b>Figure 1:</b>
+                                  <b>Figure 4:</b>
                                        Western blot of all produced and purified proteins.
                                    </p>
                                </div>
                                 </div>
-                 <p><b>Fig.&nbsp;1</b> shows that the band of the CP-LPETGG is can be seen at approximately 49&nbsp;kDa. Consequently, the successful production
+                 <p><b>Fig.&nbsp;4</b> shows that the band of the CP&#8209;LPETGG is can be seen at approximately 49&nbsp;kDa. Consequently, the successful production
-                     was proven. CP-LPETGG was detected with Strep-Tactin-HRP.</p>
+                     was proven. CP&#8209;LPETGG was detected with Strep&#8209;Tactin&#8209;HRP.</p>
-                  </p>
                   <h4>Forming multimers</h4>
-                  <p>The SDS&#8209;PAGE (<b>Fig. 2</b>)
+                  <p>The SDS&#8209;PAGE (<b>Fig. 5</b>)
 <!-- WELCHE SDS-PAGE? -->
@@ Line 218: / Line 258: @@
 , even if it is in high concentration.
                   <div style="text-align: center;">
-                         <a href="https://2019.igem.org/wiki/images/f/ff/T--TU_Darmstadt--Coat_%2B_BSA.png"target="_blank">
+                         <a href="https://2019.igem.org/wiki/images/f/ff/T--TU_Darmstadt--Coat_%2B_BSA.png" target="_blank">
                          <img class="img-fluid center" src="https://2019.igem.org/wiki/images/f/ff/T--TU_Darmstadt--Coat_%2B_BSA.png" style="max-width:60%" />
                          </a>
                          <div class="caption">
                             <p>
-                            <b>Figure 2:</b>
+                            <b>Figure 5:</b>
-                                 SDS-PAGE of BSA and CP-LPETGG.
+                                 SDS-PAGE of BSA and CP&#8209;LPETGG.
                              </p>
                          </div>
                          </div>
-                     <b>Fig.&nbsp;2</b> shows the bands of BSA at approximately 66&nbsp;kDa and the CP-LPETGG at approximately 49&nbsp;kDa. When the proteins were combined,
+                        <p>
-                     two bands can be seen, one of CP-LPETGG and one of BSA. Hence, no multimers were formed.
+                     <b>Fig.&nbsp;5</b> shows the bands of BSA at approximately 66&nbsp;kDa and the CP-LPETGG at approximately 49&nbsp;kDa. When the proteins were combined,
+                     two bands can be seen, one of CP&#8209;LPETGG and one of BSA. Hence, no multimers were formed.
                      </p>
                   <h4>Sortase-medited Reaction</h4>
-                  <p>The possibility of modification was shown with a SDS-PAGE, which shows GGGG-mCherries linked to several CP-LPETGG.
+                  <p>The possibility of modification was shown with a SDS-PAGE, which shows GGGG&#8209;mCherries linked to several CP&#8209;LPETGG.
                          <div style="text-align: center;">
-                                 <a href="https://2019.igem.org/wiki/images/e/e5/T--TU_Darmstadt--EnzymeSubstrate3.png"target="_blank">
+                                 <a href="https://2019.igem.org/wiki/images/e/e5/T--TU_Darmstadt--EnzymeSubstrate3.png" target="_blank">
                                  <img class="img-fluid center" src="https://2019.igem.org/wiki/images/e/e5/T--TU_Darmstadt--EnzymeSubstrate3.png" style="max-width:60%" />
                                  </a>
                                  <div class="caption">
                                     <p>
-                                    <b>Figure 3:</b>
+                                    <b>Figure 6:</b>
                                     SDS-PAGE of CP-LPETGG modified with mCherry by Sortase&nbsp;A7M and Sortase&nbsp;A5M.
                                      </p>
                                  </div>
                                  </div>
+<p>
-                     The SDS-PAGE shows multiple bands (<b>Fig.&nbsp;3</b>), which relate to a higher molecular weight than mCherry or CP-LPETGG themselves have.
+                     The SDS-PAGE shows multiple bands (<b>Fig.&nbsp;6</b>), which relate to a higher molecular weight than mCherry or CP&#8209;LPETGG themselves have.
-                     The bands located between 55&nbsp;kDa and 70&nbsp;kDa most likley show the linked CP-LPETGG and GGGG-mCherry, as we expected the product to be approximately
+                     The bands located between 55&nbsp;kDa and 70&nbsp;kDa most likley show the linked CP&#8209;LPETGG and GGGG&#8209;mCherry as we expected the product to be approximately .... kDa.      <!-- WELCHE GROESSE HATTEN WIR ERWARTET????   -->
-XY&nbsp;kDa.      <!-- WELCHE GROESSE HATTEN WIR ERWARTET????   -->
@@ Line 277: / Line 315: @@
-                      <sup id="cite_ref-9" class="reference">
+                      <sup id="cite_ref-10" class="reference">
-                             <a href="#cite_note-9">[9]
+                             <a href="#cite_note-10">[10]
                               </a>
                           </sup>
-                          <sup id="cite_ref-10" class="reference">
+                          <sup id="cite_ref-11" class="reference">
-                                 <a href="#cite_note-10">[10]
+                                 <a href="#cite_note-11">[11]
                                   </a>
                               </sup>
@@ Line 290: / Line 328: @@
-and  the P22 Coat Protein accommodates a cysteine residue.
+and  the P22 Coat Protein accommodates a cysteine residue (<b>Fig. 7</b>).
                       <div style="text-align: center;">
-                             <a href="https://static.igem.org/mediawiki/parts/c/c7/T--TU_Darmstadt--CP-LPETGG_Sortase.png"target="_blank">
+                             <a href="https://static.igem.org/mediawiki/parts/c/c7/T--TU_Darmstadt--CP-LPETGG_Sortase.png" target="_blank">
                          <img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/c/c7/T--TU_Darmstadt--CP-LPETGG_Sortase.png" style="max-width:60%" />
                          </a>
                          <div class="caption">
                             <p>
-                            <b>Figure 2:</b>
+                            <b>Figure 7:</b>
                                  SDS-PAGE of sortase-mediated linkage between several coat proteins with LPETGG-tag.
                              </p>
                          </div>
                          </div>
-                  </p>
-                  <p> Want to know more about sortase-mediated ligation? Please have a look at our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/Sortase"target="_blank">wiki</a>.</p>
+                  <p> Want to know more about sortase-mediated ligation? Please have a look at our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/Sortase" target="_blank">wiki</a>.</p>
                  <h4> Assembly</h4>
@@ Line 309: / Line 347: @@
                  </p>
                  <h5><i>In vivo assembled VLPs</i></h5>
-                 <p>For extracting the VLPs, which consits of SP and with sfGFP modified CP-LPETGG, directly from cell broth we first
+                 <p>For extracting the VLPs, which consits of SP and with sfGFP modified CP&#8209;LPETGG, directly from cell broth we first
                      lysed the cells by sonication and got rid of debris by two
                      centrifugation steps at 12,000&nbsp;x&nbsp;g. Afterwards ultracentrifugation with a sucrose cushion (35%&nbsp;w/v) at 150,000&nbsp;x&nbsp;g was
@@ Line 316: / Line 354: @@
                      <div style="text-align: center;">
-                     <a href="https://2019.igem.org/wiki/images/5/54/T--TU_DARMSTADT--UZ_invivo_2.png"target="_blank">
+                     <a href="https://2019.igem.org/wiki/images/5/54/T--TU_DARMSTADT--UZ_invivo_2.png" target="_blank">
                          <img class="img-fluid" src="https://2019.igem.org/wiki/images/5/54/T--TU_DARMSTADT--UZ_invivo_2.png"
                            style=max-width:40%;>
@@ Line 323: / Line 361: @@
                        <div class="caption">
                          <p>
-                           <b> Figure 3:</b>
+                           <b> Figure 8:</b>
-                           Cell broth after ultracentrifugation. Supernatant containing sfGFP-SP and CP while
+                           Cell broth after ultracentrifugation. Supernatant containing sfGFP&#8209;SP and CP while
                            VLPs collected in the sediment</p>
                        </div>
                      </div>
-                 </p>
                  <p>Ultracentrifugation sediment most likely still contains monomeric proteins and small amounts of cell debris that can
                      be harmful in some applications due to high endotoxin levels. For getting rid of these
-                     contaminants we subsequently used size-exclusion chromatography (SEC) (Sephadex-100 column).
+                     contaminants we subsequently used size-exclusion chromatography (SEC) (Sephadex&#8209;100&nbsp;column).
-                     <sup id="cite_ref-11" class="reference">
+                     <sup id="cite_ref-12" class="reference">
-                             <a href="#cite_note-11">[11]
+                             <a href="#cite_note-12">[12]
                               </a>
                      </sup>
@@ Line 342: / Line 380: @@
                       contaminants. Chromatography dilutes the sample significantly which is not optimal for analytic purposes. This is why
                       a second ultracentrifugation treatment would be required for re-concentration of purified capsids as
-                      <sup id="cite_ref-11" class="reference">
+                      <sup id="cite_ref-12" class="reference">
-                             <a href="#cite_note-11">[11]
+                             <a href="#cite_note-12">[12]
                               </a>
                      </sup>  suggest.
                       <div style="text-align: center;">
-                      <a href="https://2019.igem.org/wiki/images/e/ee/T--TU_DARMSTADT--TEM_SEC.png"target="_blank">
+                      <a href="https://2019.igem.org/wiki/images/e/ee/T--TU_DARMSTADT--TEM_SEC.png" target="_blank">
                          <img class="img-fluid" src="https://2019.igem.org/wiki/images/e/ee/T--TU_DARMSTADT--TEM_SEC.png"
                            style=max-width:60%;>
@@ Line 355: / Line 393: @@
                        <div class="caption">
                          <p>
-                           <b> Figure 4:</b>
+                           <b> Figure 9:</b>
                            Intact P22-VLPs after size exclusion chromatography</p>
                        </div>
                      </div>
-                 </p>
                  <h5><i>in vitro</i> assembled VLPs</h5>
                  <p> The images of ultracentrifugation show that monomeric proteins were separated from assembled capsids by
@@ Line 368: / Line 406: @@
                          intact VLPs all over the sample, measuring a diameter of 60&nbsp;nm or less (<b>Fig.&nbsp;2</b>).
                          <div style="text-align: center;">
-                          <a href="https://2019.igem.org/wiki/images/5/52/T--TU_DARMSTADT--invitro_UZ_TEM.png"target="_blank">
+                          <a href="https://2019.igem.org/wiki/images/5/52/T--TU_DARMSTADT--invitro_UZ_TEM.png" target="_blank">
                          <img class="img-fluid center" src="https://2019.igem.org/wiki/images/5/52/T--TU_DARMSTADT--invitro_UZ_TEM.png" style="max-width:60%" />
                          </a>
                          <div class="caption">
                             <p>
-                            <b>Figure 2:</b> Ultracentrifugation of <i>in vitro</i>assembled VLPs
+                            <b>Figure 10:</b> Ultracentrifugation of <i>in vitro</i>assembled VLPs
                              </p>
                             </div>
                          </div>
-                 </p>
                   <p> The images taken via TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. Therefore,
                       the CPs produced using this part must be fully functional. The CPs assemble with
-                      SPs and can be modified on the surface (<b>Fig.&nbsp;4</b>). Moreover, CPs also assemble without SPs
+                      SPs and can be modified on the surface (<b>Fig.&nbsp;10</b>). Moreover, CPs also assemble without SPs
-                      (<b>Fig. =&nbsp;3</b>).
+                      (<b>Fig. =&nbsp;11</b>).
                   </p>
                   <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">
                       <img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/b/bc/T--TU_Darmstadt--TEM_CP_ohne_SP.jpeg" style="max-width:40%" />
                         </a>
                          <div class="caption">
                             <p>
-                            <b>Figure 3:</b>
+                            <b>Figure 11:</b>
                                  Assembly of only coat proteins with a LPETGG-tag.
                              </p>
                          </div>
                   </div>
-                     <p><b>Fig.&nbsp;3</b> shows that no scaffold proteins are necessary for assembly.</p>
+                     <p><b>Fig.&nbsp;11</b> shows that no scaffold proteins are necessary for assembly.</p>
                      <div style="text-align: center;">
-                             <a href="https://static.igem.org/mediawiki/parts/b/b7/T--TU_Darmstadt--TEM_CP_SP_sGFP.jpeg"target="_blank">
+                             <a href="https://static.igem.org/mediawiki/parts/b/b7/T--TU_Darmstadt--TEM_CP_SP_sGFP.jpeg" target="_blank">
                      <img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/b/b7/T--TU_Darmstadt--TEM_CP_SP_sGFP.jpeg" style="max-width:40%" />
                          </a>
                          <div class="caption">
                             <p>
-                            <b>Figure 4:</b>
+                            <b>Figure 12:</b>
                                  Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.
                              </p>
@@ Line 409: / Line 447: @@
                      </div>
-                     <p><b>Fig.&nbsp;4</b> shows that CP-LPETGG and SPs assemble to VLPs and that CP-LPETGG can be modified for this process.</p>
+                     <p><b>Fig.&nbsp;12</b> shows that CP-LPETGG and SPs assemble to VLPs and that CP-LPETGG can be modified for this process.</p>
+                    <p>For more information about VLP&nbsp;assembly, please
+                            visit our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP" target="_blank">wiki</a>. </p>
-                     <p>The diameter of VLPs consisting of different protein combinations was measured with dynamic&nbsp;light&nbsp;scattering&nbsp;(DLS) analysis.
+                     <p>The hydrodynamic diameter of VLPs consisting of different protein combinations was measured with dynamic&nbsp;light&nbsp;scattering&nbsp;(DLS) analysis.
+                            In general hydrodynamic diameters depend on
+                            several properties like polarity and charges as well as size
+                            and shape. These properties can be summed up as the  electrical properties of the system.
+                            <sup id="cite_ref-13" class="reference">
+                              <a href="#cite_note-13">[13] </a> </sup
+                            >.
+                    </p>
+                    <div style="text-align: center;">
+                    <a
+                            href="https://2019.igem.org/wiki/images/9/90/T--TU_DARMSTADT--Hydro_radius.png"
+                            target="_blank"
+                          >
+                            <img
+                              class="img-fluid abstand"
+                              src="https://2019.igem.org/wiki/images/9/90/T--TU_DARMSTADT--Hydro_radius.png"
+                              style="max-width:60%;"
+                            />
+                          </a>
+                          <!--Hydrodynamischer Radius-->
+                          <div class="caption">
+                            <p>
+                              <b> Figure 13:</b>
+                              Influence of particle charge on hydrodynamic diameter.
+                            </p>
+                          </div>
                          <div style="text-align: center;">
-                                 <a href="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png"target="_blank">
+                                 <a href="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png" target="_blank">
                          <img class="img-fluid center" src="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png" style="max-width:70%" />
                              </a>
                              <div class="caption">
                                 <p>
-                                <b>Figure 5:</b>
+                                <b>Figure 14:</b>
                                     Diagram of DLS measurment of VLPs .
                                  </p>
                              </div>
                          </div>
+<p>
                          We showed by dynamic
                          light scattering (DLS) analysis (<b>Fig.&nbsp;5</b>) that capsids containing only CP are smaller than P22-VLPs containing both CP and SP. This was
                          also confirmed by measuring VLPs and CP-only capsids in TEM images using ImageJ. Capsids which are only composed of CP measured
-                         average diameter of 53nm±4.3nm are significantly smaller than VLPs out of SP and CP measured average diameter of 57nm±3nm
+                         average diameter of 53&nbsp;nm±4.3&nbsp;nm are significantly smaller than VLPs out of SP and CP measured average diameter of 57&nbsp;nm±3&nbsp;nm
                          (n=20; p&nbsp;<&nbsp;0.005). What also became clear is that the presence of the LPETGG tag does not affect the size of the assembled CP-only
                          capsid.
@@ Line 433: / Line 498: @@
                      </p>
-                     <p>For more information about VLP&nbsp;assembly, please
+                     <p>
-                             visit our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP"target="_blank">wiki</a>. </p>
+                            When we started to compare <b>sfGFP-modified VLPs</b> with
+                            <b>non-modified</b> VLPs using <b>dynamic light scattering</b> (DLS), we
+                            expected a difference in hydrodynamic radii because surface
+                            modifications should further increase the hydration of the
+                            particles as shown in
+                            <b>Fig. 3</b><sup id="cite_ref-14" class="reference">
+                              <a href="#cite_note-14">[14] </a>
+                            </sup>.
+                            <div style="text-align: center;">
+                            <a
+                            href="https://2019.igem.org/wiki/images/5/58/T--TU_DARMSTADT--DLS_VLPs_vs_VLPs.png"
+                            target="_blank"
+                          >
+                            <img
+                              class="img-fluid center"
+                              src="https://2019.igem.org/wiki/images/5/58/T--TU_DARMSTADT--DLS_VLPs_vs_VLPs.png"
+                              style="max-width:70%;"
+                            />
+                          </a>
+                          <!--DLS mit allen-->
+                          <div class="caption center">
+                            <p>
+                              <b> Figure 15:</b>
+                              Dynamic light scattering analysis. Hydrodynamic diameters of
+                              different P22-VLP species.
+                            </p>
+                          </div>
+                           <p> As described
+                            <a
+                              href="https://2019.igem.org/Team:TU_Darmstadt/Safety"
+                              target="_blank"
+                              >here</a>, non-modified VLPs showed hydrodynamic diameters of
+                            approximately 112.4&nbsp;nm&nbsp;&plusmn;&nbsp;41.3&nbsp;nm. In comparison,
+                            modified capsids showed an average hydrodynamic diameter of
+&nbsp;nm.  In our case, the drastically elevated hydrodynamic diameter of the
+                            P22-VLP linked to sfGFP may result from strong hydration since
+                            wild type sfGFP is multiply negatively charged
+                            <sup id="cite_ref-15" class="reference">
+                              <a href="#cite_note-15">[15] </a>
+                            </sup>. This probably leads to a tremendous charge density all over the surface.
+                            Another possible reason could be the formation of sfGFP dimers
+                            attached to the VLPs.
+                          </p>
+                        </div>
+                          <p>
+                            In order to demonstrate the integrity of our modified VLPs we
+                            used capsids from the same sample for DLS and electron
+                            microscopy which confirms the presence of intact VLPs. The
+                            size distribution shows that they still pose a monodisperse
+                            species, even though their hydrodynamic diameter is increased compared to
+                            unmodified VLPs or capsids containing only CP.
+                          </p>
+                        <p>For more information about VLP&nbsp;assembly, please
+                             visit our <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/VLP_Modification" target="_blank">wiki</a>. </p>
                      <h2>References</h2>
@@ Line 488: / Line 613: @@
                                                              Encapsulation Inside the Capsid of the Bacteriophage P22, American Chemical Society, 2012, 6:
 -5009
-                                                      <a rel="nofollow" class="external autonumber" href="https://pubs.acs.org/doi/pdf/10.1021/nn300545z"target="_blank">[5] </a>
+                                                      <a rel="nofollow" class="external autonumber" href="https://pubs.acs.org/doi/pdf/10.1021/nn300545z" target="_blank">[5] </a>
                                                      </span>
                                                  </li>
@@ Line 499: / Line 624: @@
                                                                  in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.,
                                                                  Biophysical Journal, 1998, 74: 559-568
-                                                          <a rel="nofollow" class="external autonumber" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299408/"target="_blank">[6] </a>
+                                                          <a rel="nofollow" class="external autonumber" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299408/" target="_blank">[6] </a>
                                                          </span>
                                                      </li>
@@ Line 510: / Line 635: @@
                              Synthetic LPETG-Containing Peptide Incorporation in the <i>Staphylococcus&nbsp;aureus</i> Cell-Wall in a Sortase&nbsp;A- and Growth
                              Phase-Dependent Manner, plos&nbsp;one, 19.02.2014
-                              <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1371/journal.pone.0089260"target="_blank">[7] </a>
+                              <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1371/journal.pone.0089260" target="_blank">[7] </a>
                              </span>
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@@ Line 521: / Line 646: @@
                                  Dustin&nbsp;Patterson, Benjamin&nbsp;LaFrance, Trevor&nbsp;Douglas, Rescuing recombinant proteins by sequestration
                                  into the P22 VLP, Chemical&nbsp;Communications, 2013, 49: 10412-10414
-                              <a rel="nofollow" class="external autonumber" href="https://pubs.rsc.org/en/content/articlelanding/2013/cc/c3cc46517a#!divAbstractcite_note-1"target="_blank">[8] </a>
+                              <a rel="nofollow" class="external autonumber" href="https://pubs.rsc.org/en/content/articlelanding/2013/cc/c3cc46517a#!divAbstractcite_note-1" target="_blank">[8] </a>
                              </span>
                          </li>
                          <li id="cite_note-9">
                                  <span class="mw-cite-backlink">
-                                    <a href="#cite_ref-9">↑</a>
+                                  <a href="#cite_ref-9">↑</a>
+                                </span>
+                                <span class="reference-text">
+                                  Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation
+                                  inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009.
+                                  <a rel="nofollow" class="external autonumber" href="https://pubs.acs.org/doi/abs/10.1021/nn300545z" target="_blank">[9]
+                                  </a>
+                                </span>
+                              </li>
+                        <li id="cite_note-10">
+                                <span class="mw-cite-backlink">
+                                    <a href="#cite_ref-10">↑</a>
                                  </span>
                                  <span class="reference-text">
                                              Jia&nbsp;X, Kwon&nbsp;S, Wang&nbsp;CI, Huang&nbsp;YH, Chan&nbsp;LY, Tan&nbsp;CC, Rosengren&nbsp;KJ, Mulvenna&nbsp;JP, Schroeder&nbsp;CI,
                                              Craik&nbsp;DJ, Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase&nbsp;A, Journal&nbsp;of&nbsp;biological&nbsp;chemistry, 2014, 289, 627-6638
-                                  <a rel="nofollow" class="external autonumber" href="http://www.jbc.org/content/289/10/6627.long "target="_blank">[9] </a>
+                                  <a rel="nofollow" class="external autonumber" href="http://www.jbc.org/content/289/10/6627.long " target="_blank">[10] </a>
                                  </span>
                              </li>
-                             <li id="cite_note-10">
+                             <li id="cite_note-11">
                                      <span class="mw-cite-backlink">
-                                         <a href="#cite_ref-10">↑</a>
+                                         <a href="#cite_ref-11">↑</a>
                                      </span>
                                      <span class="reference-text">
@@ Line 543: / Line 679: @@
                                              Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium
                                              Actinomyces oris, Journal&nbsp;of&nbsp;biological&nbsp;chemistry, 2015, 290, 21393-21405
-                                      <a rel="nofollow" class="external autonumber" href="http://www.jbc.org/content/290/35/21393.long"target="_blank">[10] </a>
+                                      <a rel="nofollow" class="external autonumber" href="http://www.jbc.org/content/290/35/21393.long" target="_blank">[11] </a>
                                      </span>
                                  </li>
-                                 <li id="cite_note-11">
+                                 <li id="cite_note-12">
                                          <span class="mw-cite-backlink">
-                                           <a href="#cite_ref-11">↑</a>
+                                           <a href="#cite_ref-12">↑</a>
                                          </span>
                                          <span class="reference-text">
                                            Patterson, D. P., Prevelige, P. E., & Douglas, T. (2012). Nanoreactors by programmed enzyme encapsulation
                                            inside the capsid of the bacteriophage P22. Acs Nano, 6(6), 5000-5009.
-                                           <a rel="nofollow" class="external autonumber" href="https://pubs.acs.org/doi/abs/10.1021/nn300545z"target="_blank">[11]
+                                           <a rel="nofollow" class="external autonumber" href="https://pubs.acs.org/doi/abs/10.1021/nn300545z">[12]
                                            </a>
                                          </span>
                                        </li>
+                                      <li id="cite_note-13">
+                                            <span class="mw-cite-backlink">
+                                              <a href="#cite_ref-13">↑</a>
+                                            </span>
+                                            <span class="reference-text">
+                                              J. Rybka, A. Mieloch, A. Plis, M. Pyrski, T. Pnioewski and
+                                              M.Giersig, Assambly and Characterization ofHBc Derived Virsus-like
+                                              Particles with Magnetic Core, Nanomaterials (Basel), 2019, 9(2):
+                                              <a
+                                                rel="nofollow"
+                                                class="external autonumber"
+                                                href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409934/"
+                                                target="_blank"
+                                                >[13]
+                                              </a>
+                                            </span>
+                                          </li>
+                                          <li id="cite_note-14">
+                                                <span class="mw-cite-backlink">
+                                                  <a href="#cite_ref-14">↑</a>
+                                                </span>
+                                                <span class="reference-text">
+                                                  https://www.horiba.com/uk/ scientific/ products/ particle-characterization/ applications/ pharmaceuticals/ viruses-virus-like-particles/
+                                                  <a
+                                                    rel="nofollow"
+                                                    class="external autonumber"
+                                                    href="https://www.horiba.com/uk/scientific/products/particle-characterization/applications/pharmaceuticals/viruses-virus-like-particles/"
+                                                    target="_blank"
+                                                    >[14]
+                                                  </a>
+                                                </span>
+                                              </li>
+                                              <li id="cite_note-15">
+                                                    <span class="mw-cite-backlink">
+                                                      <a href="#cite_ref-15">↑</a>
+                                                    </span>
+                                                    <span class="reference-text">
+                                                      Laber, J. R., Dear, B. J., Martins, M. L., Jackson, D. E.,
+                                                      DiVenere, A., Gollihar, J. D., ... & Maynard, J. A. (2017). Charge
+                                                      shielding prevents aggregation of supercharged GFP variants at
+                                                      high protein concentration. Molecular pharmaceutics, 14(10),
+-3280.
+                                                      <a
+                                                        rel="nofollow"
+                                                        class="external autonumber"
+                                                        href="https://www.ncbi.nlm.nih.gov/pubmed/28870080"
+                                                        target="_blank"
+                                                        >[15]
+                                                      </a>
+                                                      </span>
                      </ol>
@@ Line 563: / Line 754: @@
      </div>
 </html>
 <!-- Add more about the biology of this part here