Part:BBa_K5366024

T7 promoter-RBS-TET-6xHis-T7 termonator

TET structural gene expression plasmid

Construction

The screened sequence TET, 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(+)-TET plasmid

Indicator

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

Result

The results from the liquid phase analysis indicated that the concentration of the TET product was approximately 0.7 times that of UxaE under the same reaction conditions. In comparison to the screening template, the enzyme activity of the wild-type strain did not yield favorable results. Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 64
  Illegal EcoRI site found at 199
  Illegal PstI site found at 103
  Illegal PstI site found at 748
  Illegal PstI site found at 1513
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 64
  Illegal EcoRI site found at 199
  Illegal NheI site found at 1564
  Illegal PstI site found at 103
  Illegal PstI site found at 748
  Illegal PstI site found at 1513
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 64
  Illegal EcoRI site found at 199
  Illegal BglII site found at 668
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 64
  Illegal EcoRI site found at 199
  Illegal PstI site found at 103
  Illegal PstI site found at 748
  Illegal PstI site found at 1513
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 64
  Illegal EcoRI site found at 199
  Illegal PstI site found at 103
  Illegal PstI site found at 748
  Illegal PstI site found at 1513
  Illegal NgoMIV site found at 795
  Illegal AgeI site found at 352
  
• 1000
  COMPATIBLE WITH RFC[1000]