Part:BBa_K1610105

pTS + RBS + yebF + ACT3m + Term

This part consists of temperature sensitive promoter (Bba_K608351), a RBS (Bba_B0034), a motor protein sequence yebF (Bba_K1610300), a coding sequence (Bba_K1610201), and a terminator (Bba_B0015). This part is used to produce and secrete a modified human antichymotrypsin, a human extracellular protein that has been given the ability to bind and inhibit Granzyme B. This circuit is temperature regulated, where environments at or above 37C will activate the promoter.

Usage and Biology

After completing our full device, and knowing that we cannot directly test the effects of ACT3m on human GzmB (we do not have the safety clearance to test human samples!), we wanted to see if our chassis was actually expressing the correct protein. We ran an SDS-PAGE gel using E. coli samples containing our full device.

We also ran a biuret protein color indicator test, which turns from blue to purple. The biuret solution turning purple indicates that proteins were secreted into the LB. The tube containing ACT3m only (without YebF) remained blue, which indicates that proteins were not found in the solution. Combined with our SDS-PAGE results, this indicates that the full device synthesized and secreted YebF+ACT3m out of the bacteria.

SDS-PAGE gel of our protein YebF+ACT3m. Bacteria cells were grown in liquid culture and ran on a gel. Lane 3: negative control (BBa_B0015); Lane 4: full construct (BBa_K1610105). The arrows show additional proteins in bacteria carrying our full GzmB inhibitor device. However, we are unsure if any of these bands are the correct protein product.

A biuret test is a simple test for proteins. Bacterial cells expressing YebF+ACT3m or ACT3m were grown in liquid culture, and spun down to separate the cells from the LB solution. Only the supernatant was transferred to a new tube. Biuret, a protein color indicator that turns from blue to purple, was added. The tube on the left contains LB isolated from our full construct (purple). The tube on the right (blue) is LB from liquid culture of bacteria carrying ACT3m only (not fused to YebF).

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
  Illegal NotI site found at 781
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1082
  Illegal BglII site found at 1913
  Illegal XhoI site found at 2288
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2214