Part:BBa_K5366023

T7 promoter-RBS-MBC-6xHis-T7 termonator

MBC structural gene expression plasmid

Construction

The screened sequence MBC, which has potential tagatose-4-epimerase activity, was sent to a company for synthesis, and the sequence was subsequently ligated into the pET-28a(+) vector. (Figure 1).

Fig. 1 Mapping of pET-28a(+)-MBC plasmid

Indicator

The pET-28a(+)-AJC7 construct was transformed into E. coli BL21 (DE3) and incubated in an inverted culture at a constant temperature of 37°C for 14 hours. Individual colonies were selected from the transformed growth, and colony PCR was performed. Single colonies with the correct bacterial plasmid were then transferred to LB medium containing kanamycin (LB Kan) for activation and amplification culturing, followed by protein purification as outlined in [Experiment]. The final concentration of fructose in the reaction system was set at 10 g/L, supplemented with 10 µL of Ni²⁺ as a catalyst. The volume of pure enzyme solution required was determined based on the protein concentration, as indicated in [Experimental]. The reaction was conducted at 70℃ for 5 hours, and the products were analyzed using high-performance liquid chromatography (HPLC) (Figure 2).

Fig. 2 Concentration of tagatose produced by MBC and UxaE under identical reaction conditions.

Result

As indicated by the results from the liquid phase analysis, the concentration of the product obtained from MBC was approximately 1.1 times higher than that of UxaE under identical reaction conditions. This improvement in product concentration suggests that the enzyme activity of the MBC wild-type strain has also been enhanced compared to the screening template.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 262
  Illegal PstI site found at 283
  Illegal PstI site found at 658
  Illegal PstI site found at 700
  Illegal PstI site found at 910
  Illegal PstI site found at 1468
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1543
  Illegal PstI site found at 262
  Illegal PstI site found at 283
  Illegal PstI site found at 658
  Illegal PstI site found at 700
  Illegal PstI site found at 910
  Illegal PstI site found at 1468
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 262
  Illegal PstI site found at 283
  Illegal PstI site found at 658
  Illegal PstI site found at 700
  Illegal PstI site found at 910
  Illegal PstI site found at 1468
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 262
  Illegal PstI site found at 283
  Illegal PstI site found at 658
  Illegal PstI site found at 700
  Illegal PstI site found at 910
  Illegal PstI site found at 1468
  Illegal NgoMIV site found at 1117
  Illegal AgeI site found at 406
  
• 1000
  COMPATIBLE WITH RFC[1000]