Part:BBa_K4183003

Used for comparison with BBa_K4183002 to test the efficiency of secreted signal peptide.

Used for comparison with BBa_K4183002 to test the efficiency after the addition of the secreted signal peptide at the N-terminal end of lacZ.

Usage and Biology

We coated the engineered EcN bacteria containing BBa_K4183003 on LB agar plates containing IPTG as well as X-Gal and incubated them overnight at 37°C. The results showed that the breakdown of X-Gal could be clearly observed around the monoclonal colonies (Figure 1), which proved that lacZ was secreted extracellularly.

Figure1. Micrographs of X-Gal disassembled on LB agar plates containing X-Gal and IPTG. (A) When the N-terminal end of lacZ did not contain kp-sp, no disassembly of X-Gal was clearly observed around the monoclonal colonies. (B) When the N-terminal end of lacZ contains kp-sp, X-Gal is clearly observed to be disassembled around the monoclonal colony, showing a blue color.

To further measure the efficiency of kp-sp transport, we measured the efficiency of lacZ in the bacteriophage and supernatant using the β-galactosidase (β-GAL) activity assay kit (Solarbio, BC2580). The results showed (Figure 3) that the enzyme activity of lacZ in the supernatant accounted for 21.93% of the overall enzyme activity, which also proved that the efficiency of kp-sp transport was about 21.93%.

Figure2. Determination of enzymatic activity of lacZ in bacteriophage and supernatant (A) Using the standards in the kit, the standard curve was measured with R2 = 0.9997. (B) The enzyme activity of lacZ in the bacterium was 31048.32061 and 8723.369848 in the supernatant, as measured using the kit and visible light spectrophotometer.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]