Part:BBa_K4195153

T7-ampC-T7t

Biology

AmpC
β-lactamase (AmpC) is a bacterial enzyme that facilitates resistance against β-lactam antibiotics by hydrolyzing the β-lactam ring, deactivating the antibiotic. It can also catalyze the hydrolysis reaction of nitrocefin, resulting in a distinct color change from yellow to red(1).
Fig. 1 The color transformation catalyzed by AmpC.

Usage and Design

β-lactamase was selected for its good performance in enhancing the report signal. We add BBa_K3222000, BBa_K4195068, BBa_B0034 and BBa_K731721 to construct the expression system and obtained the composite BBa_K4195153, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Characterization

1. In Vivo Verification

1) Agarose Gel Electrophoresis
After transferring the plasmid into BL21(DE3), colony PCR was used to certify the plasmid was correct. The expected result was obtained.
Fig. 2 The result of colony PCR. Plasmid pSB1C3.
2) Absorbance measurement
Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of AmpC is observed by measuring the absorbance in 482 nm as time progressed using microplate reader.
Results are documented in related pages: BBa_K4195156, BBa_K4195157, BBa_K4195158, BBa_K4195160, BBa_K4195161, BBa_K4195164, BBa_K4195165, BBa_K4195166.

2. In Vitro Verification

Plasmid was put into the cell-free system for expression. The expression behavior of AmpC is observed by measuring the absorbance in 482 nm as time progressed using microplate reader.
Results are documented in related pages: BBa_K4195159, BBa_K4195162, BBa_K4195163.

Reference

1. K. E. Boehle, C. S. Carrell, J. Caraway, C. S. Henry, Paper-Based Enzyme Competition Assay for Detecting Falsified β-Lactam Antibiotics. ACS Sens 3, 1299-1307 (2018).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1237
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 38
  Illegal XhoI site found at 379
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]