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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 1000
  COMPATIBLE 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..

Induction Fermentation Protocol

(1) Co-transform the pYB1C-Gatz-PGP plasmid into E. coli BW25113. Select colonies from the validation plate and inoculate them into 5 mL liquid LB medium. Incubate at 37°C for 12 hours. (2) Transfer 50 µL of the culture into 5 mL ZYM5052 medium, and induce expression by adding appropriate concentrations of L-arabinose and IPTG. Incubate the cultures at 30°C for 18 hours, and measure the OD600 values. (3) Transfer an equal amount of cells into 200 µL M9 medium containing 20 g/L glucose, adjust the cell density, and ferment at 30°C for 12 hours.

Concentration Measurement

(1) Collect samples from the shaker and centrifuge at 13,000 rpm for 10 minutes. (2) Dilute the samples 5-fold by adding 100 µL of the sample to 400 µL of distilled water. (3)Mix the solution thoroughly. (4)Add 500 µL of resorcinol solution (0.75 g/mL) to the diluted sample. (5)Incubate the mixture at 100°C for 20 minutes. (6) Cool the samples at 4°C for 5 minutes. (7)Transfer 200 µL of the cooled mixture to a 96-well plate. (8)Measure absorbance at 405 nm using a microplate reader. (9)Record and analyze the results. This version refines clarity and enhances the technical tone to sound more professional

Figure 3:Changes in D-tagatose production in strains overexpressing gatz and pgp genes compared to strain BW25113.