1. Laboratory safety training
2. Configure LB medium
3. PCR amplification of tnaC and amilGFP template DNA
4. Inoculate pTrc99K and pET28a-inaK-gldh containing strains
5. gel extraction

1. Plasmid pTrc99K and pET28a-inaK-gldh were extracted
2. SLC7A5 gene fragments amplification
3. Gel electrophoresis
4. Gel extraction of tnaC-amilGFP, SLC7A5 gene fragments
5. Double-enzyme digestion of Overlap's large segment
6. extraction of the digested fragments
7. ligate the DNA fragments by T4 DNA ligase and transform them into Top10 competent cells

1. Transformants were identified by colony PCR
2. Select monoclonal and culture it in liquid LB medium

1. Plasmids extraction and verification by double-enzyme digestion
2. Send the correct plasmids to the company for Sanger sequencing

1. Transform the pET28a-inaK-gldh plasmid to BL21 (DE3) and incubate at 37℃.
2. Inoculate the pTrc99k-tnaC-amilGFP transformants in LB(Amp+) liquid medium overnight at 37℃, 220rpm.

1. Inoculate the pET28a-inaK-gldh plasmid containing BL21 (DE3) monoclonal in 10 mL LB(Kan+) liquid medium and culture at 37℃, 220rpm for 12h.
2. Transfer the cultured medium of pTrc99k-tnaC-amilGFP into several bottoms of LB medium with different concentrations of tryptophan, and take 200μL samples at different times.

1. Transfer the cultured medium of pET28a-inaK-gldh into fresh LB(Kan+) liquid medium and incubate at 37℃, 220rpm until OD600 is around 0.6, add IPTG to induce protein expression.
2. Measure the fluorescence of pTrc99k-tnaC-amilGFP samples.
3. Purification of the inaK-gldh protein through the Ni-NTA method.
4. Prepare SDS-PAGE protein gel and protein expression detection.

1. Glutamatase activity detection
2. Analyze and organize data