Difference between revisions of "Part:BBa K883001"

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===Results===
 
===Results===
  
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To show that this part can be used to produce wildtype CCMV VLPs, we took EM pictures of formed VLPs and measure the size distribution in the DLS.
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In order for us to be a good and functional part, we want to know if VLPs are formed with this brick using the protocol from above. We used two methods that is described ....
  
 
[[Image:CCMVWT.jpg|500px|center|thumb|''Figure 1: Wildtype CCMV VLPs taken with electron microscope'']]
 
[[Image:CCMVWT.jpg|500px|center|thumb|''Figure 1: Wildtype CCMV VLPs taken with electron microscope'']]

Revision as of 08:04, 26 September 2012

CCMV wt coat protein under IPTG induced promoter

CCMV is an abriviation of the Cowpea Chlorotic Mottle Virus, one of the most studied viruses in producing Virus Like Particles (VLPs). This part is the coding sequence of the coat protein of the CCMV virus. When expressed in Escherichia coli, these coat proteins can be harvested and be assembled in vitro using the Wageningen_UR iGEM 2012 protocols. This part is provided for the iGEM community so that they can use this part to produce these VLPs for their one own use, including but not limited to packaging, making fusion proteins to the N/C terminal. This all for to be used in a wide variety of applications. for ideas what kind of applications can be mediated with CCMV VLPs, check http://2012.igem.org/Team:Wageningen_UR/Applications


Background

This part is made from the wild type CCMV coat protein. CCMV is the abbreviation of Cowpea Chlorotic Mottle Virus, and is a well studied black eyed pea infecting virus. 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 first and best studied Virus Like Particles.


Usage and Biology

Protocols for production and purification:

  • [http://2012.igem.org/Team:Wageningen_UR/Protocol/StartupCCMV| CCMV production protocol part 1]
  • [http://2012.igem.org/Team:Wageningen_UR/Protocol/DialysisCCMV| CCMV production protocol part 2]
  • [http://2012.igem.org/Team:Wageningen_UR/Protocol/RoundupCCMV| CCMV purification protocol]


Results

In order for us to be a good and functional part, we want to know if VLPs are formed with this brick using the protocol from above. We used two methods that is described ....

Figure 1: Wildtype CCMV VLPs taken with electron microscope
Graph 1: The size distribution according to the DLS of the wildtype CCMV VLPs.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 368
  • 1000
    COMPATIBLE WITH RFC[1000]

Functional Parameters


To test the stability of the CCMV VLPs, we treat the VLPs with certain conditions. One think we want to know is, does our VLPs withstand the temperature of a ill human being. To test this we put the CCMV VLPs for 24 hours in waterbaths with different temperatures. These temperatures range from 5 degrees celcius to 40 degrees representing the ill human being. After the treatment we tested the samples under the DLS and EM.

Graph 2: The temperature stability measurements of the wildtype CCMV VLPs
figure 2: Wildtype CCMV VLPs after 24h heat treatment, taken with the EM

As seen in graph 1 and figure 1, the particles are still detected after a 24 hour treatment with the waterbaths. This shows that the particles can stay stable under temperature conditions of a ill human being.


CCMV VLPs are renown for its wide pH stability. It is stable between a pH range of 4.7 and 6.5. At a pH of 4.7 the CCMV is in it's unswollen form with a radius of around 14 nm. At a pH of 6.5 it is in it's swollen form, the VLPs radius is around 15-16nm. It is possible to change form, but change the pH gradually with the use of dialysis. If you change the pH too sudden, the VLPs are falling apart and forms aggregates as seen in figure 3.

figure 3: Wildtype CCMV VLPs aggregates after a sudden change of pH, taken with the EM


To test the stability of the VLPs during a long period of time, we deviced a experiment. We produced multiple CCMV VLPs and measure them after 3, 6 and 24 hours after formation.

Graph 3: The temperature stability measurements of the wildtype CCMV VLPs

In theory, under perfect conditions (pH at 4.7, T = 4 degrees Celcius) it is possbile to store the CCMV VLPs for a good few weeks. Be aware that aggregate formation may occure when the VLPs are exposed to changing pH or temperature.

*Safety notice*

This part is isolated from a virus and, when assembled, will form particles that very much resemble this virus in size and shape. However, no additional viral information is stored in this part and viral infection and/or replication can therefore be ruled out. It is completely safe for use in normal laboratory circumstances.