1.Add New basic part
Usage and Biology:
In the current literature, the DAEase of Thermoclostridium caenicola was expressed in Escherichia coli system, and the enzymatic properties of DAEase were further studied. DAEase is also used to convertD-fructose to D-psicose in the Co2+, because DAEase is strictly dependent on metals. The novel T. caenicola DAEase displayed maximum activity at pH 7.5 and 65 °C in the presence of 1 mM Co2+. It was strictly metal-dependent, and the addition of Co2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t1/2 value at 55 °C and 4.8 °C increase in Tm.Chen, etc.2021.
Experimental data:
In order to construct the plasmid pPICZαA-TcDAE, the target gene TcDAE was amplified by PCR.In figure 1, it is clear to see that our sample are mainly in the range between 750 bp and 1000bp, which accurately matched with theoretical length, indicating a successful Polymerase chain reaction.
Figure.1 The gel electrophoresis of TcDAE nucleic acids
Usage and Biology:
Thermogutta terrifontis was characterized as the first thermophilic representative of the phylum Planctomycetes. This bacteria have first been isolated in hot springs in Russia. As other cultivated planctomycetes, T. terrifontis grew well on various carbohydrates including oligo- and polysaccharides.(M, Garcia-Lopez, etc. 2015) . Hongbin Q studied TtDAE from Thermogutta terrifontis with a half-life of 32 h at 70°C, but did not study its yield. (Hongbin Q, etc. 2023)
Experimental data:
Figure 2. The gel electrophoresis of TtDAE nucleic acids.
In order to construct the plasmid pPICZαA-Tt DAE, the target gene TtDAE was amplified by PCR.In figure 2, it is clear to see that our sample are mainly in the range between 750bp and 1000bp, which accurately matched with theoretical length, indicating a successful Polymerase chain reaction.
Experimental data:
Figure 3. The gel electrophoresis Validation of NtDAE nucleic acids
In order to construct the plasmid pPICZαA-NtDAE, the target gene NtDAE was amplified by PCR.In figure 3, it is clear to see that our sample are mainly in the range between 750bp and 100bp, which accurately matched with theoretical length, indicating a successful Polymerase chain reaction.
Experimental data:
Figure 4. The gel electrophoresis Validation of DsDAE nucleic acids.
In order to construct the plasmid pPICZαA-DsDAE, the target gene DsDAE was amplified by PCR.In figure 4, it is clear to see that our sample are mainly in the range between 750bp and 100bp, which accurately matched with theoretical length, indicating a successful Polymerase chain reaction.
2.Add new composite part
Engineering Principle:
Psicose 3-epimerase (DAE) plays an important role in the biotransformation of D-psicose.Under the assistance of metal ions, it can convert D-fructose to D-psicose.DAE from different sources has different enzyme activity characteristics, so the yield and conversion rate of D-psicose are different under different temperature conditions. DAE derived from Thermoclostridium caenicola/Thermogutta terrifontis/Novibacillus thermophilus/Dorea sp. CAG 317 has the characteristics of heat resistance, so we will use fructose as the substrate, add the purified enzyme, and generate D-psicose under the catalysis of 40-80 degrees and Co2+.We further verified the expression of the protein and the yield of D-psicose(Figure 5).
Figure 5.The engineering schematic diagram of the project design
2.1 pPICZαA-TcDAE(BBa_K5528005)
Construction Design
Selection of novel DAEs was conducted in NCBI database. The genes encoding DAE fused with 6×His-tag at its C-terminus were optimized , and chemically synthesized.The plasmid map was constructed by snapgene software(Figure 6). and inserted into the expression vector pPICZαA between restriction endonucleases EcoRI and SalI sites, the recombinant plasmids pPICZαA-TcDAE were further transformed into E.Coli DH5α.
Figure 6. The plasmid map of pPICZαA-TcDAE
Experimental Approach
We transferred the plasmid pPICZαA-TcDAE into E.coli-DH5α and did single clone verification. Figure 7-A shows the PCR products were all highlighted between 750 and 1000, which was consistent with the length of the target gene. Figure 7-B, showed the E. coli plates we cultured and the location of sample for single clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 7-C showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing to confirm its sequence fidelity, and the positive recombinant plasmids were named as pPICZαA-TcDAE (Figure 7).
Figure 7. The single clone verification and sequencing comparison of plasmid pPICZαA-TcDAE ( E.coli DH5α )
2.2 pPICZαA-TtDAE(BBa_K5528006)
Construction Design
Selection of novel DAEs was conducted in NCBI database. The genes encoding DAE fused with 6×His-tag at its C-terminus were optimized , and chemically synthesized.The plasmid map was constructed by snapgene software(Figure 8). and inserted into the expression vector pPICZαA between restriction endonucleases EcoRI and SalI sites, the recombinant plasmids pPICZαA-TtDAE were further transformed into E.Coli DH5α.
Figure 8. The plasmid map of pPICZαA-TtDAE
Experimental Approach
We transferred the plasmid pPICZαA-TtDAE into E.coli-DH5α and did single clone verification.Figure 9-A shows the PCR products were all highlighted between 750 and 1000, which was consistent with the length of the target gene. Figure 9-B, showed the E. coli plates we cultured and the location of sample for single clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 9-C showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing to confirm its sequence fidelity, and the positive recombinant plasmids were named as pPICZαA-TtDAE (Figure 9).
Figure 9. The single clone verification and sequencing comparison of plasmid pPICZαA-TtDAE ( E.c oli DH5α )
2.3 pPICZαA-NtDAE(BBa_K5528007)
Construction Design
Selection of novel DAEs was conducted in NCBI database. The genes encoding DAE fused with 6×His-tag at its C-terminus were optimized , and chemically synthesized.The plasmid map was constructed by snapgene software(Figure 10). and inserted into the expression vector pPICZαA between restriction endonucleases EcoRI and SalI sites, the recombinant plasmids pPICZαA-NtDAE were further transformed into E.Coli DH5α.
Figure 10. The plasmid map of pPICZαA-NtDAE
We transferred the plasmid pPICZαA-NtDAE into E.coli-DH5α and did single clone verification. Figure 11-A shows the PCR products were all highlighted between 750 and 1000bp, which was consistent with the length of the target gene. Figure 11-B, showed the E. coli plates we cultured and the location of sample for single clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 11-C showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing to confirm its sequence fidelity, and the positive recombinant plasmids were named as pPICZαA-NtDAE (Figure 11).
Experimental Approach
Figure 11. The single clone verification and sequencing comparison of plasmid pPICZαA-NtDAE ( E.coli DH5α)
2.4 pPICZαA-DsDAE(BBa_K5528008)
Construction Design
Selection of novel DAEs was conducted in NCBI database. The genes encoding DAE fused with 6×His-tag at its C-terminus were optimized , and chemically synthesized.The plasmid map was constructed by snapgene software(Figure 12). and inserted into the expression vector pPICZαA between restriction endonucleases EcoRI and SalI sites, the recombinant plasmids pPICZαA-DsDAE were further transformed into E.Coli DH5α.
Figure 12. The plasmid map of pPICZαA-DsDAE
Experimental Approach
We transferred the plasmid pPICZαA-DsDAE into E.coli-DH5α and did single clone verification. Figure 13-A shows the PCR products were all highlighted between 750 and 1000bp, which was consistent with the length of the target gene. Figure 13-B, showed the E. coli plates we cultured and the location of sample for single clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 12-C showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing to confirm its sequence fidelity, and the positive recombinant plasmids were named as pPICZαA-DsDAE (Figure 13).
Figure 13. The single clone verification and sequencing comparison of plasmid pPICZαA-DsDAE ( E.coli DH5α)