Revision as of 14:50, 27 September 2024

T7 promoter-RBS-UxaE-6xHis -T7 termonator

UxaE structural gene expression plasmid

Construction

The screened sequence UxaE, 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.

Indicator

The pET-28a(+)-UxaE 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°C for 5 hours, and the products were analyzed using high-performance liquid chromatography (HPLC) (Figure 1).

< img class="bild" src="https://static.igem.wiki/teams/5366/part/wt-concentration-of-product-tagatose.png">

Sequence and Features