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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.  
 
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.  
 
<h1>Indicator</h1>
 
<h1>Indicator</h1>
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The pET-28a(+)-UxaE construct was transformed into <i>E. coli</i> 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).
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The pET-28a(+)-UxaE construct was transformed into <i>E. coli</i> 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 Ni<sup>2+</sup> 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).
 
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   < img class="bild" src="https://static.igem.wiki/teams/5366/part/wt-concentration-of-product-tagatose.png">
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   <img class="bild" src="https://static.igem.wiki/teams/5366/part/wt-concentration-of-product-tagatose.png"><br>
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  <i><b> Fig. 1 Concentration of product Tagatose<br><br></b></I>
 
   <div class="unterschrift"><bFig. 1 Construction of pMTL-Pfba-Bs2 recombinant plasmid</b>
 
   <div class="unterschrift"><bFig. 1 Construction of pMTL-Pfba-Bs2 recombinant plasmid</b>
 
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   Fig. 1 Concentration of product Tagatose
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Latest revision as of 16:02, 30 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).


Fig. 1 Concentration of product Tagatose


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1516
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 540
    Illegal BglII site found at 1302
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 294
  • 1000
    COMPATIBLE WITH RFC[1000]