Difference between revisions of "Part:BBa K5366054"
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<h1>Indicator</h1>
 
<h1>Indicator</h1>
 
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).
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<h1>Result<h1>
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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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  <img class="bild" src="https://static.igem.wiki/teams/5366/part/bbb.png"><br>
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  <i><b> 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<br><br></b></I>
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  <div class="unterschrift"><bFig. 1 Construction of pMTL-Pfba-Bs2 recombinant plasmid</b>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 14:28, 29 September 2024


T7 promoter-RBS-AJC7/S125D-6xHis-T7 termonator

AJC 7 single-point mutation expression plasmid

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<h1> 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
    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]