Difference between revisions of "Part:BBa K5366036"
| Line 30: | Line 30: | ||
<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 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). | 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). | ||
| + | <h1>Result</h1> | ||
| + | The catalytic efficiency of the L140P mutant, when subjected to a 5-hour reaction at 70°C, showed minimal improvement compared to the wild type under the same reaction conditions and substrate concentration. | ||
<html> | <html> | ||
<style> | <style> | ||
| Line 41: | Line 43: | ||
</p> | </p> | ||
</html> | </html> | ||
| − | |||
| − | |||
<!-- 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 17:57, 30 September 2024
AJC7/L140P
AJC7 single point mutant
Construction
Primers were designed for the L140P 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 L140P mutant, when subjected to a 5-hour reaction at 70°C, showed minimal improvement compared to the wild type under the same reaction conditions and substrate concentration.
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
- 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]