Plasmid

Part:BBa_K5024008

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


pGEX-4T-1-UAA

pGEX-4T-1-UAA


BBa_K5024001 to BBa_K5024008

BBa_K5024001 (allantoinase)

Profile

Name: allantoinase

Base Pairs: 1377 bp

Origin: Animal allantoinase is derived from kidney, liver and other tissues, Synthetic

Properties: Allantoinase is an enzyme mainly involved in the reaction of uric acid metabolic pathway. The allantoinase can convert allantoin into another metabolite-allantoic acid.

Usage and Biology

Allantoinase is used in the medical field to study and treat uric acid-related diseases, such as gout. By regulating the activity of allantoinase in the uric acid metabolic pathway, it can help reduce uric acid levels1. And allantoinase is a good candidate material for the development of allantoin biosensors2. In addition, allantoinase plays an important role in the field of plants. It is involved in the regulation of uric acid metabolism pathway and nitrogen metabolism in plants. Related research can help improve crop yield, stress resistance and nutritional efficiency3-4.

Reference:

  1. A. I R,Ali H,Sunyoung K, et al. E. coli allantoinase is activated by the downstream metabolic enzyme, glycerate kinase, and stabilizes the putative allantoin transporter by direct binding[J]. Scientific Reports,2023,13(1).
  2. Biosensors; Study Data from M. Marchetti et al Provide New Insights into Biosensors (Immobilization of Allantoinase for the Development of an Optical Biosensor of Oxidative Stress States)[J]. Biotech Week,2020.
  3. Ignacio C L,Carolina M,Alejandro C G, et al. Allantoin accumulation mediated by allantoinase downregulation and transport by Ureide Permease 5 confers salt stress tolerance to Arabidopsis plants.[J]. Plant molecular biology,2016,91(4-5).
  4. Peng W,Huang C. Allantoinase and dihydroorotase binding and inhibition by flavonols and the substrates of cyclic amidohydrolases[J]. Biochimie,2014,101.

Basic Part

BBa_K5024002 (allantoicase)

Profile

Name: allantoicase

Base Pairs: 1035 bp

Origin: Neodiprion lecontei, Synthetic

Properties: Allantoicase is an allantoicase-degrading enzyme. Allantoin is an organic compound mainly found in the allantoic sac of animal embryos.

Usage and Biology

Allantoicase can be used to analyze and study the allantoicase metabolic pathway, and to understand its function and regulatory mechanism in vivo 1-2.

Reference:

  1. H O,P T,Y I. Fibrin membrane endowed with biological function. V. Multienzyme complex of uricase, catalase, allantoinase and allantoicase.[J]. Biochimica et biophysica acta,1980,611(1).
  2. J E G,D G V,C D D V. Hydrolysis, racemization and absolute configuration of ureidoglycolate, a substrate of allantoicase.[J]. Biochimica et biophysica acta,1970,198(3).

Composite Part

BBa_K5024008 (pGEX-4T-1-UAA)

Construction Design

BBa_K5024008 (pGEX-4T-1-UAA) is composed of BBa_K5024000(Uric acid oxidase), BBa_K5024001(allantoinase), BBa_K5024002(allantoicase), and BBa_K5024006 (pGEX-4T-1).

To convert to Nissle1917, we replaced pET28a with pGEX-4T-1. The pGex-4T-1 is chosen for its property of functioning stably in Nissle 1917. The next step is to construct the plasmids that are directly used for target proteins synthesis in following processes. At the same time, the GST tag of pGEX4T-1 can be replaced with His. We replaced the GST tag on pGEX-4T-1 with His by homologous recombination. We use homologous recombination to assemble allantoinase and allantoicase into pGEX-4T-1-UAA.

The plasmid map of pGEX-4T-1-UAA

Figure 1. The plasmid map of pGEX-4T-1-UAA

Engineering Principle

The aim is to reduce the concentration of uric acid without causing significant negative side effects to the patient. Studies have shown that some animals have the ability to use uric acid oxidase to convert uric acid into allantoin, helping to reduce the accumulation of uric acid in their bodies.

Human urea metabolism process

Figure 2. Human urea metabolism process

Experimental Approach

  1. The construction of pGex-4T-1-UAA plasmid
    The length of the target gene allantoinase is 1485 bp. The length of the target gene allantoicase is 1074 bp. Figure 3A shows that the fragment lengths are consistent with the results. The length of the plasmid pGex-4T-1-UAO is 5301 bp. Figure 3B shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the target gene.
    Electrophoresis for the genes allantoinase, allantoicase and pGex-4T-1-UAO

    Figure 3. Electrophoresis for the genes allantoinase, allantoicase and pGex-4T-1-UAO

    Next, we transformed the plasmid pGex-4T-1-UAA into DH5α. Figure 4AB shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene allantoinase is 1485 bp. Figure 4C shows that the fragment lengths are consistent with the results. In Figure 4D, line1,4,5 shows that the allantoicase band is approximately 1024bp. It indicates that we have successfully translated the plasmid pGex-4T-1-UAA to E.coil DH5α.
    The electrophoresis of the monoclonal antibody in E.coil DH5α

    Figure 4. The electrophoresis of the monoclonal antibody in E.coil DH5α

    Afterward, we chose positive isolated colonies 1, 4, and 5 and sent them to the company for sequencing. The results showed that the sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid was normal, without any genetic mutations. It indicates that our plasmid pGex-4T-1-UAA has been successfully constructed.
    The sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid

    Figure 5. The sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid

  2. Protein expression
    In order to improve the expression of the protein, we first tested the protein in E.coil BL21(DE3). So we transformed the plasmid pGex-4T-1-UAA into E.coil BL21(DE3). Figure 6A and B show that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene allantoinase is 1485 bp and allantoinase is 1047bp. Figure 6 C and D show that the vector pGex-4T-1-UAA was successfully transformed into E.coil BL21(DE3).
    The electrophoresis of the monoclonal antibody for pGex-UAA in BL21(DE3)

    Figure 6. The electrophoresis of the monoclonal antibody for pGex-UAA in BL21(DE3)
    Note:
    Figure 6A-B: The monoclonal plate pGex-UAA in BL21(DE3)
    Figure 6C: The Electrophoresis verification of allantoicase in pGex-UAA (BL21(DE3))
    Figure 6D: The Electrophoresis verification of allantoinase in pGex-UAA (BL21(DE3))

    In order to improve the expression of UAA protein, we designed different gradients of IPTG induction. The size of the UAA protein is 118.6 kDa. Figure 7 shows that the crude protein has the target protein UAA. However, 0.1mmol, 0.25 mmol, 0.5mmol ,0.7mmol and 1mmol IPTG had no significant effect on the expression of UAA protein.
    The pGex-4T-1-UAA of SDS-PAGE in E.coil BL21(DE3)

    Figure 7. The pGex-4T-1-UAA of SDS-PAGE in E.coil BL21(DE3)

Characterization/Measurement

A. Enzyme activity detection

  1. The urate oxidase activity of UAA
    In order to determine the ability of our constructed plasmid pGex-4T-1-UAA to decompose uric acid, we used uric acid as a substrate and added UAO protein. Table 1 and Figure 8 showed that with the increase of UAA protein concentration, the content of uric acid gradually decreased, indicating that UAA is active and can successfully decompose uric acid. And we used E.coil BL21 (DE3) as the control group, the uric acid content of the control group had no obvious change trend.
    The curve of UAA activity

    Figure 8. The curve of UAA activity

  2. Contrast enzyme activity of UAO and UAA
    By comparing the levels of uric acid after adding UAA and UAO proteins (Figure 9), it was found that the uric acid content was lower with UAA compared to UAO. This suggests that UAA has slightly higher enzymatic activity than UAO, further indicating that UAA has a stronger ability to degrade uric acid. However, the protein expression level of UAA can be optimized in order to enhance its enzymatic activity in subsequent experiments.
    The curve of UAA and UAO activity

    Figure 9. The curve of UAA and UAO activity

Reference:

T. J. Major, N. Dalbeth, E. A. Stahl, T. R. Merriman, An update on the genetics of hyperuricaemia and gout. Nat Rev Rheumatol 14, 341-353 (2018)



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 956
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 930
    Illegal BamHI site found at 8117
    Illegal XhoI site found at 951
    Illegal XhoI site found at 6606
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2089
    Illegal SapI.rc site found at 342
    Illegal SapI.rc site found at 3171


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