Part:BBa_K5366055
T7 promoter-RBS-AJC7/T181A-6xHis-T7 termonator
AJC 7 single-point mutation expression plasmid
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).
Result
Under the reaction conditions of 70℃ for 5 hours, the catalytic efficiency of the T181A mutant was improved compared to the wild type. However, this enhancement was not particularly pronounced, even though it was statistically significant under the same substrate concentration and conditions.
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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1552
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 540
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1042
- 1000COMPATIBLE WITH RFC[1000]
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