Part:BBa_K1689012

dCas9-Nlact

N terminal of β-lactamase fused with dCas9

β-Lactamase (AmpR) is produced by some bacteria, providing resistance against penicilins and so on. The β-Lactamase hydrolyzes β-lactam's conservative ring region, deactivating the antibiotic, and it can be use for the treatment of bacterial infection.
In our project, β-Lactamase is used to catalyze the hydrolysis of penicillin to penicillinoic acid. This redox reaction leads to current changes, which can be detected using A3 electrode system [1]. We dissected β-Lactamase between Gly196 and Leu198 [2], for they are on the opposite side of the active site. Moreover, we can enhance the activity and metabolically stability of β-Lactamase by introducing a mutation M182T, which disrupts an inactive molten-globule intermediate of β-Lactamase.



Reference:
1. do Prado, T. M., Foguel, M. V., Gonçalves, L. M., & Maria del Pilar, T. S. (2015). β-Lactamase-based biosensor for the electrochemical determination of benzylpenicillin in milk. Sensors and Actuators B: Chemical, 210, 254-258.
2. Shekhawat, S. S., & Ghosh, I. (2011). Split-protein systems: beyond binary protein–protein interactions. Current opinion in chemical biology, 15(6), 789-797.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1150
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4
  Illegal BamHI site found at 3429
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]