Difference between revisions of "Part:BBa K2276003"

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T7 promoter and the coding sequneces of serotonin N-acetyltransferase (SNAT) with N-His tag

Biobrick BBa_K2276003 is a composite, consisting of T7 promotor, 6xHis tag, thrombin site, and the coding sequence of serotonin N-acetyltransferase (SNAT).

Sequence and Features

Usage and Biology:

Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. It is found in all living organisms including bacteria, insects, fungi, animals and plants that have been examined. As we all know, melatonin plays pleiotropic roles ranging from antioxidant activity to a diverse array of biological functions in animals and plants [1]. For example, The Nobel Prize in Physiology or Medicine 2017 was awarded jointly to Jeffrey C. Hall, Michael Rosbash and Michael W. Young "for their discoveries of molecular mechanisms controlling the circadian rhythm". [2]. And melatonin participates in the regulation of circadian rhythms in mammals. For its important functions, many efforts spared to find out the biosynthetic pathway of melatonin. In fact, two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Fortunately, the SNAT genes, the product either from an animal or a plant, were functionally soluble in E.coli. However, to co-fuction with the other three enzymes, especially the enzyme SNAT, we must choose one that can guarantee the final expression of melatonin arrive at a high level. So, we followed the research [3] and chose the SNAT comes from sheep to express the enzyme in our E.coli BL21 (DEC) system.

Results:

Link the SNAT the N-terminal His tag:

In our experiments, we want to figure out and confirm the function of SNAT. In other words, only we obtained the complete pure enzymes, we could find out its function. Thus, firstly we linked SNAT with the vector pET15(+), which has N-terminal His tag that make the protein purification easier. And this work was verified by bacterial colony PCR (Figure.1).

Confirm the SNAT was expressed successfully in E.coli BL21( DE3):

When the plasmid was constructed successfully, we must detect whether the enzyme we obtained can express in E.coli system. Therefore, we transferred the pET15- SNAT plasmid into E.coli BL21 (DE3) strain [3] for expression. What’s more, in order to find out where the protein exactly exists, we used the SDS-PAGE to detect the different compositions of the bacteria lysate (Figure.2).

Prove the SNAT really function in E.coli system:

However, there was still a doubt that if the enzyme really works in the E.coli BL21 (DE3) system. Hence, in order to verify the SNAT enzyme catalyzes the reaction to convert tryptophan into tryptamine, we used the HPLC (High Performance Liquid Chromatography), a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture, to test the reduction of the substrate and the increment of the product. We did this by two ways: in vivo, we added the substrate tryptophan into the culture media. After a period of culturing, we tested the tryptophan content in the media and the product content in bacterial lysate (Figure.3). For another thing, in vitro, we lysed the bacteria at first, and then added the substrate and test the content of it and the product respectively. By the HPLC experiment, we confirmed the SNAT enzyme really works in our E.coli BL21 (DE3) system.

Reference

[1] Arnao MB, Hernández-Ruiz J (2014) Melatonin: plant growth regulator and/or biostimulator during stress? Trends Plant Sci 19:789–797

[2] Emery P, Stanewsky R, Hall J C, et al. Drosophila cryptochromes: A unique circadian-rhythm photoreceptor[J]. Nature, 2000, 404(6777):456-457.

[3] Byeon Y, Back K. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase[J]. Applied Microbiology & Biotechnology, 2016, 100(15):6683.

[4] VoisinP, Namboodiri MA, KleinDC (1984) Arylamine N-acetyltransferase andarylalkylamine N-acetyltransferase in the mammalian pineal gland. J Biol Chem 259:10913–10918

[5] Vriend J, Reiter RJ (2015) Melatonin feedback on clock genes: a theory involving the proteasome. J Pineal Res 58:1–11

[6] KangK, LeeK, ParkS, KimYS ,BackK(2010) Enhanced production of melatonin by ectopic overexpression of human serotonin N-acetyltransferase plays a role in cold resistance in transgenic rice seedlings. J Pineal Res 49:176–182

[7] Kang K, Lee K, Park S, Byeon Y, Back K (2013) Molecular cloning of rice serotonin N-acetyltransferase ,the penultimate gene in plant melatonin biosynthesis. J Pineal Res 55:7–13

[8] Kang K, Kong K, Park S, Natsagdorj U, Kim YS, Back K (2011) Molecular cloning of a plant N-acetylserotonin methyltransferase and its expression characteristics in rice. J Pineal Res 50: 304–309