Difference between revisions of "Part:BBa K5366037"

 
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AJC7 two-point mutant
 
AJC7 two-point mutant
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<h1>Construction</h1>
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The mutation site of T181A is located far from S125D, allowing for the direct construction of the S125D/T181A double mutant using pET-28a(+)-AJC7-S125D as a template along with the primers designed for single-point mutation as outlined in [Design 1]. Following the mutation, the PCR products were verified using nucleic acid gel electrophoresis. The plasmids that exhibited the correct bands were subsequently transformed into E. coli BL21 (DE3) competent cells.
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  <img class="bild" src="https://static.igem.wiki/teams/5366/part/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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  <div class="unterschrift"><bFig. 1 Construction of pMTL-Pfba-Bs2 recombinant plasmid</b>
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  <img class="bild" src="https://static.igem.wiki/teams/5366/part/nucleic-acid-gel-plot-of-colony-pcr.png"><br>
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  <i><b> Fig. 2 nucleic-acid-gel-plot-of-colony-pcr<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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<h1>Indicator</h1>
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The two-point 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 described in [Experimental]. The volume of the purified enzyme solution required for the 500 μL reaction system was determined based on the protein concentration indicated in [Experimental]. The final concentration of fructose in the system was set at 100 g/L, with the addition of 10 μL of Ni²⁺ as a catalyst. The reaction was conducted at 70°C for 5 hours, after which the products were analyzed using High-Performance Liquid Chromatography (HPLC).
 
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===Usage and Biology===
 
===Usage and Biology===

Revision as of 12:55, 28 September 2024


AJC7/S125D/ T181A

AJC7 two-point mutant

Construction

The mutation site of T181A is located far from S125D, allowing for the direct construction of the S125D/T181A double mutant using pET-28a(+)-AJC7-S125D as a template along with the primers designed for single-point mutation as outlined in [Design 1]. Following the mutation, the PCR products were verified using nucleic acid gel electrophoresis. The plasmids that exhibited the correct bands were 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 two-point 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 described in [Experimental]. The volume of the purified enzyme solution required for the 500 μL reaction system was determined based on the protein concentration indicated in [Experimental]. The final concentration of fructose in the system was set at 100 g/L, with the addition of 10 μL of Ni²⁺ as a catalyst. The reaction was conducted at 70°C for 5 hours, after which the products were analyzed using High-Performance Liquid Chromatography (HPLC). 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]