Difference between revisions of "Part:BBa K5366033"

 
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AJC7 single point mutant
 
AJC7 single point mutant
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<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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  <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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  <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/fig-2-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 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).
 
<!-- 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 12:15, 28 September 2024


AJC7/T181A

AJC7 single point mutant

Construction

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 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). 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]