Part:BBa_K5366025

T7 promoter-RBS-HDM-6xHis -T7 termonator

HDM structural gene expression plasmid

Construction

The screened sequence HDM, 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(+)-HDM plasmid.

Indicator

The pET-28a(+)-HDM 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 HDM and UxaE under identical reaction conditions.

Result

The results from the liquid phase analysis indicate that the concentration of the HDM product was approximately two times higher than that of UxaE under the same reaction conditions. This suggests that the activity of HDM has also improved compared to the screening template strain.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1555
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]