Team:Wageningen UR/ModifyingtheCCMV

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== Part 1: Modifying the CCMV VLP for site specific drug delivery ==
 
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==== Introduction ====
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= Cowpea Chlorotic Mottle Virus =
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CCMV is the abbreviation of Cowpea Chlorotic Mottle Virus, and is a well studied black eyed pea infecting virus (figure 1). It is a virus in the family Bromoviridae, and it is one of the most studied plant viruses. As from the discovery of the virus in 1967, it was an interesting virus due to the easy cultivation and harvesting procedures involved. Interest in this virus is especially in the ability to disassemble the virus, remove the content and refold the empty particle simply by altering the pH. This makes this virus one of the best studied Virus-like Particles.
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[[File:cowpeadisease.jpg|500px|center|thumb|<p align="justify">''Figure 1: Symptoms caused by [http://www.ces.ncsu.edu/depts/pp/notes/Soybean/soy009/soy009.htm CCMV]</p>]]
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==VLP==
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The idea of disassembly and reassembly of CCMV into VLPs did not stop by the formation of empty particles. Little inorganic crystals and proteins were also able to be captured into the shell. The production of these VLPs can also be integrated into a synthetic biology approach. Expression of only the coat protein in ''Escherichia coli'' or yeast could also provide the coat proteins that are able to assemble and reassemble when playing with the pH (figure 2). This method opened the interest of a whole new kind of [https://2012.igem.org/Team:Wageningen_UR/Applications applications] for these VLPs, naming medical applications. This is because this synthetic approach removes the pathogenic factor, the virus, from the production process. This assists in the acceptance of these kind of particles in the application of treating (plant)-diseases.
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[[File:1cwp.PNG|500px|center|thumb|<p align="justify">''Figure 2: 3d model of [http://viperdb.scripps.edu/info_page.php?VDB=1cwp CCMV]</p>]]
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CCMV will be one of the VLPs that we will be working on. We will focus on this VLP to prove the concept of using it as a Drug Delivery System.
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==Methods==
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'''Aim''': Producing CCMV VLPs with epitopes on the outside or packaging coils in the inside, with the possibility to combine both to deliver a bio Nano carrier, with ligands on the outside and “medicine” packed on the inside.
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The template of the CCMV coat protein was provided by the Virology group Wageningen, in a pET28 vector, a normal ''E. coli'' expression vector. We used primers that already included the prefix and suffix of Standard 10, so that after amplification, the gene can be directly ligated into a pSB1C3 backbone. It also allows the combination with an inducible promoter and RBS directly. In order to make the modified VLPs, we designed primers with an overhang containing the modifications. A detailed description of the primers and the modifications can be found in the [[Team:Wageningen_UR/Journal|Journal]].
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==== Outside modification of CCMV VLP ====
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== Results ==
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To aid in a better and quicker response to newly emerging pathogens, a more standardized approach of vaccine production could help in containing pandemic outbreaks. Faster reaction in vaccine development and production could be achieved with a universal approach and giving vaccine developers the opportunity to adapt their process better to the demand using the newly developed Quality by Design approach. The focus will be on providing a universal approach for a connector system. The aim is to create a “Plug 'N Apply System". The concept is that only the epitopes need to be selected and produced, after which they can be applied upon one standard vaccination platform. These epitopes are fused to connector peptides. The production will be one standardized synthetic biology procedure, and produced in a single, production friendly organism. The connector peptide is able to “plug onto” a standardized platform, which then provides an expression surface ready for immunization of subjects. The standardized platform can be produced as a bulk reservoir to provide massive reserves of backbone for a sudden explosive demand of vaccine. The industry can shift their production according to the demand by solely selecting the epitope that they fuse to the connector. This will provide more flexibility and quicker response to emerging pathogenic threats
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We isolated the CCMV coat protein coding sequence from an expression plasmid obtained from Dr. Kormelink (Virology group Wageningen UR) and ligated it into a standard 10 backbone. With an IPTG promotor biobrick part (BBa_J04500) from the registry we made an IPTG inducable CCMV coat protein expression biobrick part. These particles are measured in the [[Team:Wageningen_UR/MethodsDetection|transmision electron microscopy]] (figure 3) and with [[MethodsDetection#Dynamic_Light_Scattering_.28DLS.29|Dynamic Light Scattering]] (graph 1).
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==== inside modification of CCMV VLP ====
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<center>
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{|class="wikitable"
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|[[File:Ccmv_EM.tif|300px|thumb|<p align="justify">''Figure 3: An image from the Transmission Electron Microscopy of CCMV VLPs produced with E.coli. An ability of the TEM is characterising the diameter of the VLPs, which is done in this image. Also an example of a wrong formation of the VLP is observed </p>]]
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|[[File:DetectionDLSCCMV.jpg|300px|thumb|''Graph 1: Size distribution according to DLS results of the wildtype CCMV VLPs.'']]
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|}
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</center>

Latest revision as of 02:28, 27 September 2012


Contents

Cowpea Chlorotic Mottle Virus

CCMV is the abbreviation of Cowpea Chlorotic Mottle Virus, and is a well studied black eyed pea infecting virus (figure 1). It is a virus in the family Bromoviridae, and it is one of the most studied plant viruses. As from the discovery of the virus in 1967, it was an interesting virus due to the easy cultivation and harvesting procedures involved. Interest in this virus is especially in the ability to disassemble the virus, remove the content and refold the empty particle simply by altering the pH. This makes this virus one of the best studied Virus-like Particles.

Figure 1: Symptoms caused by [http://www.ces.ncsu.edu/depts/pp/notes/Soybean/soy009/soy009.htm CCMV]

VLP

The idea of disassembly and reassembly of CCMV into VLPs did not stop by the formation of empty particles. Little inorganic crystals and proteins were also able to be captured into the shell. The production of these VLPs can also be integrated into a synthetic biology approach. Expression of only the coat protein in Escherichia coli or yeast could also provide the coat proteins that are able to assemble and reassemble when playing with the pH (figure 2). This method opened the interest of a whole new kind of applications for these VLPs, naming medical applications. This is because this synthetic approach removes the pathogenic factor, the virus, from the production process. This assists in the acceptance of these kind of particles in the application of treating (plant)-diseases.

Figure 2: 3d model of [http://viperdb.scripps.edu/info_page.php?VDB=1cwp CCMV]

Methods

The template of the CCMV coat protein was provided by the Virology group Wageningen, in a pET28 vector, a normal E. coli expression vector. We used primers that already included the prefix and suffix of Standard 10, so that after amplification, the gene can be directly ligated into a pSB1C3 backbone. It also allows the combination with an inducible promoter and RBS directly. In order to make the modified VLPs, we designed primers with an overhang containing the modifications. A detailed description of the primers and the modifications can be found in the Journal.

Results

We isolated the CCMV coat protein coding sequence from an expression plasmid obtained from Dr. Kormelink (Virology group Wageningen UR) and ligated it into a standard 10 backbone. With an IPTG promotor biobrick part (BBa_J04500) from the registry we made an IPTG inducable CCMV coat protein expression biobrick part. These particles are measured in the transmision electron microscopy (figure 3) and with Dynamic Light Scattering (graph 1).

Figure 3: An image from the Transmission Electron Microscopy of CCMV VLPs produced with E.coli. An ability of the TEM is characterising the diameter of the VLPs, which is done in this image. Also an example of a wrong formation of the VLP is observed

Graph 1: Size distribution according to DLS results of the wildtype CCMV VLPs.
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