Part:BBa_K883161

CCMV_Negative core antigen with IPTG induced promotor

This construct produces CCMV core antigen subunits. The subunits have been modified in such a way that they will fold into CCMV Virus-Like Particles that have a negative interior. This was done by replacing 6 Arginine and 3 Lysine residues at the N-Terminus with Glutamic acid residues. The promotor and RBS that have been added to the gene allow induced expression of the subunits which increases the yield. The full protocol for this construct can be found on the [http://2012.igem.org/Team:Wageningen_UR Wageningen_UR 2012 Wiki].

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 the EM to take pictures of the formed VLP.

EM picture of a CCMV VLP with an negetivly charged interior

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 370
  
• 1000
  COMPATIBLE WITH RFC[1000]

*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.