Part:BBa_K5453005
pBAD-Gatz-PGP-Trrnb
Gatz and PGP were successively ligated under the promoter of pBAD, and the expression of the corresponding proteins was induced after the addition of arabinogalactan inducer, so as to construct the Tagatose synthesis pathway in E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 658
Illegal NgoMIV site found at 784
Illegal AgeI site found at 329
Illegal AgeI site found at 1556 - 1000COMPATIBLE WITH RFC[1000]
Design
Since Escherichia coli lacks the pathway for synthesizing D-tagatose, we synthesized the gatz gene from Caldilinea aerophila and the pgp gene from Archaeoglobus profundus, and constructed the gatz and pgp genes into the pYB1c vector to obtain the pYB1c-Gatz-PGP plasmid.
Figure 1:Schematic diagram of the pYB1c-Gatz-PGP plasmid.
Experiments
We first amplified the gatz and pgp fragments and purified the amplified products through gel extraction. Then, using the Gibson assembly technique, we ligated the gatz and pgp fragments into the pYB1c vector. After transforming the assembled products into DH5α competent cells, we performed colony PCR, restriction enzyme analysis, and sequencing validation, ultimately successfully constructing the pYB1c-Gatz-PGP plasmid.
Figure 2:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for pYB1c, Gatz, and PGP. B: Gel electrophoresis image of colony PCR performed on 10 randomly picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI and EcoRI enzymes. D: Sequencing of the correct plasmids verified by colony PCR and enzyme digestion..
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