Part:BBa_K4291008

T7 pro- His tag-Lac operate-T7 tag-inaK-gldh-T7 ter

Profile

Name: T7 pro- His tag-Lac operate-T7 tag-inaK-gldh-T7 ter

Base Pairs: 1485 bp

Origin: Pseudomonas syringae and E.coli

Properties: a tool to display the glutamate dehydrogenase on the surface of bacteria

Usage and biology

Glutamate dehydrogenase (gldh) is a mitochondrial enzyme that is involved in the metabolism of glutamate to 2-oxoglutarate, and reversibly converts glutamate to α-ketoglutarate as part of the urea cycle. The gldh enzyme is found primarily in the liver, kidney, and cardiac muscle, while the liver has the highest concentration of gldh activity and lower levels in the brain, skeletal muscle, and leukocytes. GLDH has a housekeeping role in cell metabolism. In addition, the bacteria such E.coli also use the NADP+-specific GLDH to disposal of inorganic nitrogen.

Construct design

Because the T7 promoter and T7 RNA polymerase have strong ability in translation and usually be used as protein expression, we chose pET28a-vector and E. coli BL21(DE3), with T7 promoter and T7 RNA polymerase respectively, to express our target protein inak-gldh. To achieve this, we optimized the DNA sequences of inak-gldh and inserted them into the HindIII and NcoI sites of the pET28a vector (Figure 1.), and transformed the recombinant plasmid into E. coli BL21(DE3) for protein expression.

Figure 1. Map of pET28a- inak-gldh plasmid

BBa_K4291006

Name: inak

Base Pairs: 537 bp

Origin: Pseudomonas syringae

Properties: carrier protein in bacterial surface display system

Usage and biology

BBa_K4291006 is an encoding sequence of ice nucleation protein (inaK), which anchors in extracellular membrane using N-terminal domain. The C-terminal is used to fused target protein such as vaccines, enzymes and viral protein which displayed on the surface of bacteria.

K4291007

Name: gldh

Base Pairs: 1260 bp

Origin: E.coli

Properties: NADP-dependent glutamate dehydrogenase

Usage and biology

BBa_K4291006 is an encoding sequence of glutamate dehydrogenase (gldh). The NADP-dependent glutamate dehydrogenase has capacity of conversion the glutamate to α-ketoglutarate and ammonia.

Experimental approach

1.1 Verification by double-enzyme digestion

To build the plasmid, we let the synthetic company synthesize the DNA fragment of optimized inak-gldh and inserted it into the HindIII and NcoI sites of the pET28a vector (Figure 2A). Then, we send it to the company for Sanger sequencing. The returned sequencing comparison results showed that there were no mutations in the gene region (Figure 2B), and the plasmid pET28a- inak-gldh was successfully constructed. And the next step was extracting the recombinant plasmid and transforming it into E. coli BL21(DE3) competent cells. meanwhile, the plasmid was extracted and performed double-enzyme digestion to conform if the bacteria contain the plasmid. After that, the bacteria was used to express the inak-gldh proteins.

Figure 2. double-enzyme digestion result

1.2 Sanger sequencing of recombinant plasmid

In addition, we sent the plasmid to the company for Sanger sequencing. The certificate of recombinant plasmid sequencing results is as Figure3.

Figure 3. sequences alignment of pET28a- inak-gldh plasmid

Proof of function

2.1 protein purification

In order to verify the inak-gldh protein expression level, we cultured pET28a- inak-gldh containing BL21(DE3) strain in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.6. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used the SDS-PAGE method to verify the expression level of the target protein (Figure 4). As a result, we could significantly find the band at the correct size.

Figure 4. SDS-PAGE

2.2 Functional test of inaK-gldh

2.2.1 Measure the standard curve of NADPH

Glutamate dehydrogenase could use L-glutamate as a substrate, with reversible oxidation and deamination under the action of coenzyme (NAD + or NADP +), and the NADH or NADPH generated by the reaction has an obvious absorption peak at 340 nm. To detect glutamate dehydrogenase activity, the amount of NADPH generated by the hydrogenase catalytic reaction was measured spectrophotometrically. Absorption values at 340 nm were determined using a UV-visible spectrophotometer (Figure 5). One unit of enzymatic activity was defined as the production of 1 μ mol of the reduced product, NADPH, per OD600 cells per minute.

Figure 5. the principle of glutamate dehydrogenase reaction

NADPH was diluted to 10 μM, 50 μM, 100 μM, 400 μM, 500 μM, and 500 μM, and the absorption peaks at 340nm at each concentration were measured by spectrophotometer. With the light absorption value of NADPH at 340 nm as the vertical coordinate and the corresponding concentration of NADPH (μM) as the abscissa, the linear fitting was performed to obtain the linear regression equation corresponding to the standard curve and the standard curve.

2.2.2 inaK-gldh enzymatic activities

Cells containing the expression vector pET28a-inaK-gldh were centrifuged and washed twice with 100mM Tris-HCl (pH 8.0) buffer after induction. The standard reaction system contained bacterial cells (OD600-1.0), 100mM Tris-HC1 buffer (pH 8.0), sodium L-glutamate (final concentration 2mM), and NADP + (final concentration 0.5mM). The reaction was performed at 60°C in a 1.5mL centrifuge tube for 2min and was terminated by centrifugation of the bacteria at 12,000 rpm for 1min. The absorptive values at 340 nm were measured using a UV-visible spectrophotometer. As a result, the enzyme activity of glutamine dehydrogenase is around 567.92975 U/mL. So that the biosensor we constructed worked well.

According to the calculation formula, we obtained the enzyme activity, shown as Table 1.The enzyme activity calculation formula is as follows:

Table 1. The result of inaK-gldh enzymatic activities

• Vsample: 0.05 mL, V total: 1×10-3 L, ε: NADH molar extinction coefficient, 6.22×103 L/mol/cm, d: Cuvette light diameter, 1 cm, ∆T: Reaction time, 2 min.

Improvement

We have already collected the figures from our experiments. Glutamate dehydrogenase plays an important role in L-glutamate metabolism. After transforming the sequence-optimized gene inaK-gldh into the host strain, we can easily detect the metabolites NADPH using our biosensor. What’s more, we measured the activity of inaK-gldh, and found that the inaK-gldh worked well in the E. coli system.

The task of developing new portable tests and effective treatments is imminent. In the future, when the biosensor is improved, it may be applied to ASD detection, which can provide a new tool for disease diagnosis, and the earlier the diagnosis is made, the better the outcome.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 161
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 573
  Illegal XhoI site found at 1344
  Illegal XhoI site found at 1386
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 759
  Illegal AgeI site found at 1263
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 945
  Illegal BsaI site found at 1194