Part:BBa_K1879001

promoter + CBZ cleavage enzyme + terminator

This part contains the CBZ-cleavage enzyme with a promoter (BBa_J23118) and RBS upstream of the coding sequence, and terminator (BBa_J61048) downstream of the sequence to make a fully functioning BioBrick. This BioBrick is used in our biocontainment system to cleave the CBZ protecting group from leucine prior to incorporation of leucine into proteins. This part can be used by other iGEM teams to selectively cleave the CBZ protecting group from amino or hydroxyl groups in organic reactions.

Usage and Biology

The functionality of this part was confirmed. The product of this part is a CBZ protecting group cleavage enzyme. To test it, we transformed this part into JW E. coli cells. This strain is a leucine auxotroph, meaning that the JW cells cannot produce its own leucine. So, we tested these cells by plating them on minimal media. On just minimal medium plates, the JW cells did not grow because no leucine was supplemented (negative control). On minimal medium + leucine plates, the cells grew because of the leucine supplement (positive control). On minimal medium + CBZ-leucine plates, the cells grew. This confirms that the cleavage enzyme was functioning correctly: it cleaved CBZ from CBZ-leucine to produce leucine. So, the leucine auxotroph cells survived. We also plated normal JW cells (no transformed cleavage enzyme) on minimal medium + CBZ-leucine as another negative control, which did not produce growth. This confirms that the cleavage enzyme is functional in our BioBrick.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1402
  Illegal AgeI site found at 354
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 56
  Illegal BsaI.rc site found at 1377