Difference between revisions of "Part:BBa K5366032"
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AJC7 single point mutant
 
AJC7 single point mutant
 
<h1>Construction</h1>
 
<h1>Construction</h1>
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Primers were designed for the T181A point mutation, and the plasmid containing the pET-28a(+) vector was amplified using PCR. Following the mutation, the PCR products were verified by nucleic acid gel electrophoresis to confirm the presence of the desired bands. The plasmid containing the correct bands was subsequently transformed into <i>E. coli</i> BL21 (DE3) competent cells.
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Primers were designed for the S125D point mutation, and the plasmid containing the pET-28a(+) vector was amplified using PCR. Following the mutation, the PCR products were verified by nucleic acid gel electrophoresis to confirm the presence of the desired bands. The plasmid containing the correct bands was subsequently transformed into <i>E. coli </i>BL21 (DE3) competent cells.
 
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   <img class="bild" src="https://static.igem.wiki/teams/5366/part/fig-1-point-mutation-localisation-and-primer-design-2.png"><br>
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   <img class="bild" src="https://static.igem.wiki/teams/5366/part/fig-1-point-mutation-localisation-and-primer-design.png"><br>
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   <i><b> Fig.1 Point mutation localisation and primer design<br><br></b></I>
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   <i><b> Fig.1 Point mutation localisation and primer design <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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The mutant and wild-type strains were subjected to activation and amplification culture, followed by a series of protein purification procedures to extract the target proteins, as outlined in [Experimental]. The volume of the purified enzyme solution needed for the 500 μL reaction system was determined based on the protein concentration detailed in [Experimental]. The final concentration of fructose in the reaction mixture was 100 g/L, and it included 10 μL of Ni<sup>2+</sup> as a catalyst. The reaction was conducted at 70°C for 5 hours, and the resulting products were subsequently analyzed using High-Performance Liquid Chromatography (HPLC).
 
The mutant and wild-type strains were subjected to activation and amplification culture, followed by a series of protein purification procedures to extract the target proteins, as outlined in [Experimental]. The volume of the purified enzyme solution needed for the 500 μL reaction system was determined based on the protein concentration detailed in [Experimental]. The final concentration of fructose in the reaction mixture was 100 g/L, and it included 10 μL of Ni<sup>2+</sup> as a catalyst. The reaction was conducted at 70°C for 5 hours, and the resulting products were subsequently analyzed using High-Performance Liquid Chromatography (HPLC).
 
<h1>Result</h1>
 
<h1>Result</h1>
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Under the reaction conditions of 70℃ for 5 hours, the catalytic efficiency of the T181A mutant was improved compared to the wild type. However, this enhancement was not particularly pronounced, even though it was statistically significant under the same substrate concentration and conditions.
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The catalytic efficiency of the S125D mutant was nearly doubled compared to the wild type when both were subjected to a 5-hour reaction at 70°C under identical reaction conditions and substrate concentrations. This finding suggests that the S125D mutation represents a very effective modification for AJC7.
 
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Revision as of 15:16, 29 September 2024


AJC7/S125D

AJC7 single point mutant

Construction

Primers were designed for the S125D point mutation, and the plasmid containing the pET-28a(+) vector was amplified using PCR. Following the mutation, the PCR products were verified by nucleic acid gel electrophoresis to confirm the presence of the desired bands. The plasmid containing the correct bands was subsequently transformed into E. coli BL21 (DE3) competent cells.


Fig.1 Point mutation localisation and primer design


Fig. 2 Nucleic acid gel plot of colony PCR

Indicator

The mutant and wild-type strains were subjected to activation and amplification culture, followed by a series of protein purification procedures to extract the target proteins, as outlined in [Experimental]. The volume of the purified enzyme solution needed for the 500 μL reaction system was determined based on the protein concentration detailed in [Experimental]. The final concentration of fructose in the reaction mixture was 100 g/L, and it included 10 μL of Ni2+ as a catalyst. The reaction was conducted at 70°C for 5 hours, and the resulting products were subsequently analyzed using High-Performance Liquid Chromatography (HPLC).

Result

The catalytic efficiency of the S125D mutant was nearly doubled compared to the wild type when both were subjected to a 5-hour reaction at 70°C under identical reaction conditions and substrate concentrations. This finding suggests that the S125D mutation represents a very effective modification for AJC7.


Fig.3 Fig.3 The concentrations of tagose produced in the system after WT, S125D, T181A, H342L, I129T, and L140P reacted with 100 g/L substrate fructose for 5 h

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 501
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1003
  • 1000
    COMPATIBLE WITH RFC[1000]