Part:BBa_K861140

GalU,glucose-1-phosphate uridylyltransferase

UTP-glucose-1-phosphate uridylyltransferase (GalU) carries out a key step in the generation of UDP-D-glucuronate.GalU catalyzes the addition of UTP to α-D-glucose 1-phosphate to yield UDP-D-glucose,which is the substrate for cellulose synthetase complex to produce cellulose.

Thinker-SC 2023

Description

As the GalU gene plays a crucial role in enhancing bacterial extracellular polysaccharide (EPS) production, we have decided to overexpress galU. EPS plays a key role in bacterial soil crust by providing microbial adhesion to soil particles and promoting microbial colonization and growth on soil surfaces. Additionally, extracellular polysaccharides can form microbial aggregates, which are of great significance for microbial survival and adaptation to the soil environment.

Usage and Biology

We plan to overexpress the galU gene using the T7 promoter in the pET23b vector. B0015 is used as an additional terminator. The constructed plasmid will then be transformed into E. coli Rosetta (host cells). A schematic diagram illustrating this process can be seen in Figure 1.

Figure 1 The design of galU.

Figure 2. Gel electrophoresis of the galU.

Characterization

The successfully engineered strain will be expanded in culture.At predetermined time points (4, 8, 12, 16 hours), bacterial growth was monitored by measuring the OD600. Concurrently, cells were centrifuged, and the supernatant was used to quantify EPS using the anthrone-sulfuric acid method. In brief, a sample of the supernatant was combined with anthrone reagent, followed by the addition of concentrated sulfuric acid. The mixture was subsequently heated, and the developed color was measured spectrophotometrically at 620 nm. A standard curve, established using known concentrations of a standard polysaccharide, facilitated the determination of the EPS concentration in the samples. The EPS concentration was then normalized to the OD600 to obtain EPS production per unit of bacterial growth. To establish a standard curve, 0-200 mM glucose was used as the standard substance and quantified using the anthrone colorimetric method, with absorbance measured at 620 nm, as shown in Figure 3A. Compared to the control group, the engineered bacteria significantly increased EPS levels, which also increased with time, as shown in Figure 3B. The results indicate that the engineered strain can significantly enhance EPS production.

Figure 3. The expression effect of galU.

All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD).

Potential application directions

This experiment has broad application potential in promoting microbial colonization and growth on soil surfaces. It can improve soil quality, enhance crop growth, increase agricultural productivity, and contribute to ecological restoration and environmental conservation.

Characterized by XJTU-China 2020

We cloned and confirmed the target fragment by PCR. The length of our fragment is 981bp. The following results showed the successful result.

Figure 1:PCR confirmation of the target fragment.

the mRNA level of B.s GalU is ~2.4 fold higher than that of E.coli, indicating the significant improvement of enzyme expression and activity. However, due to the lower expression of another key enzyme PGM for EPS production in BE strain, the final EPS yield is lower than that of EE strain but still comparable. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 69
  Illegal AgeI site found at 387
  Illegal AgeI site found at 510
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 783