TU Darmstadt

Proof of concept

Introduction

We, the iGEM team TU Darmstadt, developed a modular Virus-like particle platform (MVP) based on the P22 Virus-like particle (VLP). With the real MVP many different applications are possible to challenge. For example to accelerate the development of VLP-based vaccines and drug-delivery systems (DDS). In order to use VLPs for these applications, proteins or molecules have to be either linked to the surface or interior of the VLPs.
The fast development process depends on an adaptable and reproducible system which can handle lots of different proteins reliably.
In order to modify VLPs we chose genetic fusion for the inside of the particles and a sortase mediated linking reaction for the surface. Through expert advice we know the importance of the modification ratio for the integrity of MVPs. We tested in vivo methods to control the modification ratio through reporter genes (sfGFP and mCherry) and developed a prototype bioreactor system for the automated in vivo production of VLPs with a specific modification ratio.
To prove the viability of “The real MVP” we demonstrated:

Successful assembly of VLPs

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Characterization of SortaseA5M and A7M

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Modification of VLPs via sortase-mediated ligation

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control of modification ratio

Successful Assembly and modification

The first step towards the MVP was to demonstrate correct VLP assembly from coat (CP)- and scaffold (SP)-protein. CP forms the outer shell, and SP stabilizes the VLP from the inside. We genetically fused sfGFP to the scaffold protein. Afterwards, we performed an in vitro assembly with purified proteins and visualy checked the success in a transmission electron microscope (TEM). The TEM showed correctly assembled VLPs and dynamic light scattering (DLS) demonstrated a specific VLP species. These experiments prove that you can load P22 VLPs with cargo.

Figure 1: Distribution of particle hydrodynamic diameters in DLS analysis. A monodisperse species of particles can be seen.

Figure 2: Left: VLPs loaded with genetically fused SP-sfGFP, right: expected pellet of VLPs, which is not visible due to the lack of fluorescence.

Figure 3: TEM image of in vitro assembled VLPs after ultracentrifugation.

Characterization of Sortase A5M and Sortase A7M

Animation 1: Obtained Sortase A7M Models in a principle component analysis. A fast (blue) and a slow (red) mode show the most prominent movements of the Cα-chain of candidate S_14771. Both modes show movement of the loop consisting of residues 105 to 115 towards the active site.

For a useful application, these VLPs have to be modified. We successfully modified tagged P22 coat proteins with Sortase A5M. We extensively characterized this Sortase, but also Sortase A7M for future in vivo VLP modification and their kinetics using SDS-PAGE, FRET experiments and in silico modeling. We proved the successful modification of the exterior of our VLPs with sfGFP via dynamic light scattering (DLS) and ultracentrifugation.

Figure 4: Sediment containing P22-VLPs modified with sfGFP using SortaseA5M. Sediment was imaged in transmission electron microscope

modification of VLPs via sortase mediated ligation

To demonstrate that we can modify VLPs using the sortase mediated ligation, we produced VLPs with the LPETGG-tag. The VLPs were then mixed with sortase and polyG-sfGFP for the linking reaction. The modified particles were then compared with unmodified particles using DLS.

Figure 5: Hydrodynamic diameter of CP-only capsids and unmodified VLPs

Control of modification ratio

Figure 6: Proof of concept calibration curve for the adjustment of VLP surface modifiction. Test of our dual expression system with two reporter genes for fluorescent proteins: Varios indication concentrations of AHT(blue) lead to this asymetric sigmoidal regression (red) of the ratio of mCherry to sfGFP. The samples were taken in triplicates after an overnight expression.

The VLP surface modification ratio is an important parameter. Which ratio is best depends on the application. In principle, it is possible to control the degree of surface modification by changing the amount of tagged and untagged CP during in vivo expression. As proof of concept, we controlled the expression ratio of two fluorescent proteins using a dual expression plasmid. In particular, we can set the desired ratio by changing the inducer concentration. We successfully tested this dual expression concept in a custom bioreactor system. This is a first step towards industrial MVP production.

Medal Criteria

Registration and Giant Jamboree Attendance: We registered for the Giant Jamboree and are looking forward to attend the Giant Jamboree. See you all there!
Competition Deliverable: We have created our wiki and are excited to present it to you. We finished our judging form which can be viewed here . During the giant jamboree we will showcase our project to you in form of a presentation and poster. We hope you’ll attend.
Attributions: To see who supported us during the year, have a look at our attributions site.
Project Inspiration and Description: Click here to see what we wanted to achieve with our project and what inspired us.
Characterization: Working with two different variants of Sortase A we took an aready existing one from iGEM Stockholm 2016 (BBa_K2144008)and characterized it further. We also further characterized a TetR repressed RFP (BBa_K577895).

Validated Part: We created, used and characterized multiple new parts for example P22 coat protein with LPETGG tag (BBa_K3187000).
Collaboration: Over the course of the year we collaborated with different teams. Our main collaboration was with iGEM team Freiburg. But you can have a look at all our collaborations here.
Human Practices: We cummunicated with the public as well as with experts. Through our Human Practices we could generate a lot of input coming from students to older generations to experts of the field for our project

Integrated Human Practices: We spoke to different groups of interests and ages, addressing their worries and incorporating their ideas in our project design. Especially, regarding the biosafety aspect of our project. If you want to know more click here.
Improve a Previous Part: We have improved the chromoprotein amilGFP (BBa_K1073024) and created a new version (BBa_K3187015) of it. We inserted a new 5' UTR which leads to an enhancing effect on the translation efficiency.
Model Your Project: For the modification of our VLPs we used the enzyme Sortase A7M which up to date has no known crystal structure. We used homology modeling to predict the structure of this specific enzyme. You can view those results amongst others here.
Demonstration of Your Work: We think our project is working under real world conditions. Why we think so, you can see on this page and on our results summary page.