Part:BBa_K5206008
PrT7-ctrpiB
The BBa_K5206007 (ribose-5-phosphate isomerase, RpiB) derived from Clostridium thermocellum was ligated to pET-28a(+) to construct the recombinant plasmid. The BBa_I719005(T7 promoter) and BBa_K731721(T7 terminator) respectively control the start and termination of transcription of this gene.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 487
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 228
- 1000COMPATIBLE WITH RFC[1000]
Result
(1)Plasmid construction
The vector and target gene fragments were amplified using primers RpiB-pd-FOR, RpiB-pd-REV and ZT-For, ZT-REV, respectively, and the recombinant plasmids were constructed using Gibson assembly.
(2)SDS-PAGE
After construction is completed, the PET-28a-CtrpiB is transferred into BL21(DE3) competent cells. The transformed strain was placed in 37℃ shaker and IPTG was added when OD600=0.6-0.8. Then it was transferred to 25℃ shaker for induction for 16-20h, the bacterial liquid was centrifuged and the bacterium was broken in order to detect the protein expression. As shown in the figure, obvious protein bands appeared at slightly more than 15 kDa, while the size of CtrpiB protein was about 16 kDa, proving that it was successfully induced.
(3)Fluorescence detection
BBa_K5206005 efficiently converts D-allulose to D-allose in a biocatalytic process, and the production of this conversion product can be accurately detected by an advanced biosensor system, thus providing a highly sensitive and selective analytical method for the biosynthesis of D-allose.
The BBa_K5206007 and the better-performing BBa_K5206005 were co-transformed into Escherichia coli BL21(DE3) to explore the effect of adding different concentrations of the substrate D-allulose on the biosensor at different temperatures.
One group of bacterial solution was always induced and cultured in a shaker at 30℃ for 18-24 hours. The other group was first induced in a shaker at 25℃ for 6-8 hours and then transferred to a shaker at 37℃ for continuous induction and culture.
The results showed that at 30℃, in the strain containing the sensor system, as the concentration of D-allulose increased, the fluorescence signal was enhanced in grades, proving the production of D-allose. When co-cultured at 25℃ and 37℃, when the concentration of D-allulose increased to 100 mM, the fluorescence signal slightly weakened.
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