Difference between revisions of "Part:BBa K5366022"
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AJC7 structural gene expression plasmid
 
AJC7 structural gene expression plasmid
 
<h1>Construction</h1>
 
<h1>Construction</h1>
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The screened sequence AJC7, 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).
+
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).
 
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<html>
 
<style>
 
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</p>
 
</p>
 
</html>
 
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   Fig. 1 Mapping of pET-28a(+)-AJC7 plasmid
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   Fig. 1 Mapping of pET-28a(+)-TET plasmid
 
<h1>Indicator</h1>
 
<h1>Indicator</h1>
 
The pET-28a(+)-AJC7 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℃ for 5 hours, and the products were analyzed using high-performance liquid chromatography (HPLC) (Figure 2).
 
The pET-28a(+)-AJC7 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℃ for 5 hours, and the products were analyzed using high-performance liquid chromatography (HPLC) (Figure 2).
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   Fig. 2 Concentration of tagatose produced by AJC7 and UxaE under identical reaction conditions.
 
   Fig. 2 Concentration of tagatose produced by AJC7 and UxaE under identical reaction conditions.
 
<h1>Result</h1>
 
<h1>Result</h1>
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From the results obtained in the liquid phase, it is evident that the concentration of tagatose produced by AJC7 is approximately four times higher than that produced by UxaE under the same reaction conditions. This indicates that AJC7 demonstrates significantly greater potential activity as a tagatose-4-epimerase, leading to its selection for subsequent studies.
+
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.
  
 
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Revision as of 15:28, 27 September 2024


T7 promoter-RBS-AJC7-6xHis -T7 termonator

AJC7 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(+)-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 AJC7 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
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1534
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 540
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1042
  • 1000
    COMPATIBLE WITH RFC[1000]