Plasmid

Part:BBa_K5024003

Designed by: Gao Zixuan   Group: iGEM23_BZK   (2023-08-07)


pET28a-UAO

pET28a-UAO


BBa_K5024003 - Composite Part (pET28a-UAO)

BBa_K5024003 - Composite Part (pET28a-UAO)

Construction Design

BBa_K5024003 (pET28a-UAO) is composed of BBa_K3521004 (pet28a-backbone) and BBa_K5024000 (Uric acid oxidase). We firstly choose pET28a as the vectors for the plasmids with target gene sections UAO. We choose pET28a for the His section it involves. The product Uric acid oxidase and pET28a vector were digested with NEB restriction enzymes BamHI and XhoI, and the vector fragment was recovered by gel recovery (Figure 1).

The plasmid of pET28-UAO

Figure 1. The plasmid of pET28-UAO.

Engineering Principle

Uric acid is a waste product of the metabolism of purines in the food; most of the uric acid is excreted through the liver. However, if the body is unable to efficiently excrete uric acid, uric acid levels will rise, forming urate crystals and depositing in the joints and surrounding soft tissue, causing a painful and inflammatory response that leads to joint pain and swelling [1,2]. Some animals use urate oxidase to catalyze the conversion of uric acid into allantoin, which helps reduce the accumulation of uric acid in the body (Figure 2). Our project aims to use probiotics to synthesize the enzymes (UAO) that can digest the uric acid to work as a cure for gout.

The engineering schematic diagram of the project

Figure 2. The engineering schematic diagram of the project.

Experimental Approach

We extract the pET28a vectors from existing bacterial solutions. Primarily using centrifuging, we extract the vectors from the bacterial solutions. The final products are obtained from the remnants in the absorption column. Then, the vectors are linearized by using restrictive enzymes. The electrophoresis is conducted to examine the products. Cyclical vectors move faster during electrophoresis than the linearized vectors.

By comparing the horizontal relationships between the stripes of target gene sections and the marker, we have concluded that in the same conditions, the pET28a control group moves faster during electrophoresis, and the pET28a samples move slower during electrophoresis. The length of the pET28a vector is 5369 bp, and the control group of Figure 3 is correct. Line 1-2 is the band of pET28a cut with BamHI and XhoI. This phenomenon in the figure indicates that the vectors pET28a in the sample groups have been linearized.

The electrophoresis for testing the linearized vector pET28a

Figure 3. The electrophoresis for testing the linearized vector pET28a.

Note: Line 1-2: The band of pET28a cut with BamHI and XhoI; Line 3-4: The band of pET28a cut with BamHI and NotI; Line 5: The band of pET28a cut with SacI and XhoI.

The lengths of gene fragments UAO are 981bp. By comparing the horizontal relationships between the stripes of target gene sections and the marker, Figure 4 concluded that the fragment lengths are consistent with the results. Therefore, the figure indicates that target genes of UAO are successfully amplified.

The electrophoresis for the target genes

Figure 4. The electrophoresis for the target genes.

Next, we connected UAO with pET28a by T4 ligase, and transformed the plasmid into DH5α. Figure 5A shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene UAO is 981 bp. Figure 5C concluded that the fragment lengths are consistent with the results. We have successfully transformed the vector pET28a-UAO into DH5α, and the sequencing results in Figure 5B showed no gene mutation. It indicates that our plasmid has been successfully constructed.

The Electrophoresis of the Monoclonal Antibody in DH5α

Figure 5. The Electrophoresis of the Monoclonal Antibody in DH5α.

Reference

[1] T. Neogi, Clinical practice. Gout. N Engl J Med 364, 443-452 (2011).

[2] Y. Zhu, B. J. Pandya, H. K. Choi, Prevalence of gout and hyperuricemia in the US general population: the National Health and Nutrition Examination Survey 2007-2008. Arthritis Rheum 63, 3136-3141 (2011).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5320
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3540
    Illegal NgoMIV site found at 3700
    Illegal NgoMIV site found at 5288
  • 1000
    COMPATIBLE WITH RFC[1000]


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