Plasmid

Part:BBa_K4929003

Designed by: Wang Jingmin   Group: iGEM23_SubCat-HongKong   (2023-08-23)


pAO815-AocI

Composite Part - BBa_K4929003(pAO815-AocI)

Composite Part - BBa_K4929003(pAO815-AocI)

Construction Design

After preparing the PCR templates for the target genes AocI(BBa_K4929002) and the linear pAO815(BBa_K4929001) vector, we performed separate PCR amplifications for these two fragments. The amplified fragment was then used to synthesize the pAO815-AocI(BBa_K4929003) plasmid. Then, the groups of plasmids were transformed into DH5α cells and lysed, followed by agarose gel electrophoresis to confirm the correctness of the resulting products. Finally, These plasmids were introduced into our yeast strain for further protein synthesis and functional verification.

Figure 1
Figure 1. Map of pAO815-AocI

Engineering Principle

This gene encodes a metal-binding membrane glycoprotein that oxidatively deaminates putrescine, histamine, and related compounds. The encoded protein is inhibited by amiloride, a diuretic that acts by closing epithelial sodium ion channels. Alternatively, spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Jan 2013]

Figure 2
Figure 2. Gene Map of AocI.

Amine oxidases (AOs) catalyze the oxidation of biogenic amines to form aldehydes, ammonia, and hydrogen peroxide. AOs were first discovered by Yamada et al [1], where fungi formed AOs in mycelium when grown with mono- or diamines as nitrogen sources, whereas other nitrogen sources did not have any effect on enzyme formation. Certain microorganisms can secrete amine oxidase, which reduces the biogenic amine content in fermented foods; meanwhile, biogenic amine oxidase, which can degrade biogenic amines, also exists in the human gut and is used to regulate intracellular amine content, maintain intra- and extracellular acid-base balance, and safeguard human health [2]. The classification of AOs mainly depends on their molecular structures, amino acid sequences, and cofactor structures. Currently, amine oxidases of microbial origin are found to be mainly flavin-containing amine oxidases (EC 1.4.3.4) and copper-containing amine oxidases (EC 1.4.3.6).

Copper amine oxidases (CuAOs) are a class of copper-containing reductases, and such enzymes contain two types of cofactors: 2,4,5-dihydroxyphenylalanine quinone (TPQ) and lysine tyrosine quinone (LTQ) that are produced post-translationally from specific tyrosine residues [3]. CuAOs are present in Escherichia coli, Arthrobacter globigii, Hansenula polymorphica Arthrobacter sphaericus, Hansenula polymorphica, and Picrosporum, and the amine oxidase from Arthrobacter sphaericus KAIT-B-007 was purified twice on a dextran gel S-200 [4]. The specific activity of amine oxidase from Arthrobacter sphaericus KAIT-B-007 was 14.3 U/mg. The enzyme has good heat resistance, and the enzyme activity remained stable at 65°C, and the residual enzyme activity was 91% after 10 min at 70°C [5]. The copper-containing oxidase 2 from Arthrobacter taurus TC-1 showed high catalytic efficiency for phenylethylamine, tyramine and histamine, and the relative value of Kcat/Km was 100:49.6:7.6 [6]; Hansenula polymorpha H525 contains a copper amine oxidase gene, and the crude enzyme solution was added to grape juice containing biogenic amines for 7 d, the content of phenylethylamine and tyramine was reduced to almost zero [4].

Figure 3
Figure 3. The successful construction of recombinant plasmid pAO815-AocI.

Experimental Approach

To construct the plasmids, we commissioned the company to generate gene templates for AocI. These gene fragments were then inserted into the pAO815 vector, a specific type of plasmid. We successfully constructed the pAO815-AocI plasmid and introduced it into Escherichia coli bacteria. After agarose gel electrophoresis and gel extraction, we transferred the plasmid into the yeast cells. Then, we confirmed the expression of our target protein, the amine oxidase, in the yeast cells. Finally, we will lyse and obtain the DH5α Escherichia coli cells containing the target plasmids. After agarose gel electrophoresis, we will recover the gel and reintroduce these plasmids into the GS115 yeast strain selected for this experiment. After a few days of cultivation, we will use SDS-PAGE gel electrophoresis to determine whether the yeast has successfully synthesized our target protein - Saccharomyces cerevisiae.

Figure 4
Figure 4. Testing the existence of AocI protein in pAO815-AocI.

Characterization/Measurement

We tested the enzyme’s function of decomposing the biogenic amine, and it turns out it worked well because there is yellow around the yeast in Figure 5.

Figure 5
Figure 5. The results of the pAO815-AocI transparent circle experiment and the control group.

In addition to this, we also performed HPLC tests. Histamine concentration was measured at five different temperatures (20,25,30,37,45) at different sampling times (12,24,36,48,72h). We first cultured the bacterial solution to OD0.6-0.8, so that the bacteria had the highest viability. Histamine was then added, and the initial concentration of histamine added was 200ug/ml. Use HPLC, that is, high-performance liquid chromatography to detect changes in histamine content. According to the results of the image, the content of histamine decreased gradually with the extension of time, and the degradation of histamine was the fastest at 30 degrees. At 72h, the histamine content was the lowest.

Figure 6
Figure 6. HPLC results of pAO815-AocI.

References

  1. Yamada H, Adachi O, Ogata K. Amine oxidases of microorganisms: part I. Formation of amine oxidase by fungi. Agricultural and biological chemistry, 1965, 29(2): 117-123.
  2. Song Y, Dong Q. Research progress in formation and control of the biogenic amine in Chinese rice wine. Science and Technology of Food Industry, 2016, 37(8): 387-391.
  3. Li BB, Lu SL. The importance of amine-degrading enzymes on the biogenic amine degradation in fermented foods: a review. Process Biochemistry, 2020, 99: 331-339.
  4. Mathias B, Urs M, Helmut K, et al. The potential of the yeast Debaryomyces hansenii H525 to degrade biogenic amines in food. Microorganisms, 2015, 3(4): 839-850.
  5. Yoshinori S, Hiroko M, Akira Y, et al. A thermostable histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007. Journal of Bioscience and Bioengineering, 2004, 97(2): 104-110.
  6. Lee JI, Kim YW. Characterization of amine oxidases from Arthrobacter aurescens and application for determination of biogenic amines. World J Microbiol Biotechnol, 2013, 29(4): 673-682.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 286
    Illegal NheI site found at 1233
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4114
    Illegal BamHI site found at 86
    Illegal BamHI site found at 8714
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3111
    Illegal NgoMIV site found at 6532
    Illegal NgoMIV site found at 7621
    Illegal AgeI site found at 52
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2573
    Illegal BsaI site found at 3421
    Illegal BsaI.rc site found at 5487
    Illegal BsaI.rc site found at 7705
    Illegal SapI site found at 4404


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