Difference between revisions of "Part:BBa K5366037"
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AJC7 two-point mutant | AJC7 two-point mutant | ||
| + | <h1>Molecular Docking</h1> | ||
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| + | <img class="bild" src="https://static.igem.wiki/teams/5366/part/docking-model-of-d-fructose-in-the-s125dt181a-mutant.png"><br> | ||
| + | <i><b> Fig.2 Docking model of D-fructose in the S125D/T181A mutant.<br><br></b></I> | ||
| + | <div class="unterschrift"><bFig. 1 Construction of pMTL-Pfba-Bs2 recombinant plasmid</b> | ||
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<h1>Construction</h1> | <h1>Construction</h1> | ||
| − | 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 <i> E. coli BL21 </I> (DE3) competent cells. | + | 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 <i> E. coli BL21 </I> (DE3) competent cells. |
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<h1>Indicator</h1> | <h1>Indicator</h1> | ||
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). | 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). | ||
| + | <h1>Result</h1> | ||
| + | The results demonstrated that the concentration of products obtained from the AJC7 enzyme after the two-point mutation of S125D/T181A, under the conditions of a 70℃ reaction for 5 hours, was significantly higher than the enzyme activity enhancement observed in the wild-type AJC7-S125D. Furthermore, the catalytic efficiency of the enzyme was enhanced by nearly two-fold compared to that of the wild-type AJC7. This indicates that the S125D/T181A mutation is an exceptionally effective double mutation for enhancing the performance of AJC7. | ||
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| + | <img class="bild" src="https://static.igem.wiki/teams/5366/part/aaa.png"><br> | ||
| + | <i><b> Fig.3 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P after reaction with 100g/L fructose substrate for 5 h, respectively<br><br></b></I> | ||
| + | <div class="unterschrift"><bFig. 1 Construction of pMTL-Pfba-Bs2 recombinant plasmid</b> | ||
| + | </div> | ||
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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=== | ||
Latest revision as of 18:12, 30 September 2024
AJC7/S125D/ T181A
AJC7 two-point mutant
Molecular Docking
Fig.2 Docking model of D-fructose in the S125D/T181A 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).
Result
The results demonstrated that the concentration of products obtained from the AJC7 enzyme after the two-point mutation of S125D/T181A, under the conditions of a 70℃ reaction for 5 hours, was significantly higher than the enzyme activity enhancement observed in the wild-type AJC7-S125D. Furthermore, the catalytic efficiency of the enzyme was enhanced by nearly two-fold compared to that of the wild-type AJC7. This indicates that the S125D/T181A mutation is an exceptionally effective double mutation for enhancing the performance of AJC7.
Fig.3 The concentrations of tagatose in wild-type, S125D, S125D/T181A, S125D/H342L, S125D/I129T, and S125D/L140P after reaction with 100g/L fructose substrate for 5 h, respectively
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 501
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1003
- 1000COMPATIBLE WITH RFC[1000]