Part:BBa_K2276005
pTet and the coding sequneces of caffeic acid o-methyltransferase (COMT) with N-His tag
Biobrick BBa_K2276005 is a composite, consisting of tet operator, 6xHis tag, thrombin site, and the coding sequence of caffeic acid O-methyltransferase (COMT).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 973
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 759
Illegal AgeI site found at 1209 - 1000COMPATIBLE WITH RFC[1000]
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. However, due to the low enzyme activity of ASMT in E.coli, we chose the caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that also has ASMT activity [3]. Actually, the enzyme COMT exists in many species, but we should choose one with high bioactivity in E.coli system to make the final product——melatonin arrive at the maximum expression level. So, The COMT we used comes from the plants genome —— rice , showing the higher bioactivity in an E.coli system than other resource. [4] But in our project, there is an important point that the COMT is the key enzyme in the biosythesized pathway. It is the rate-limiting enzyme at the last step. So in order to couple with the cycling-repressing system, we choose added the tet operator (because in this way of combination, the whole system will be most stabilized) front of the COMT enzyme. In this way the expression of the COMT can be controlled by our repressilators. Finally, its expression level can change periodically.
Results:
Link the COMT with N-terminal His tag and tet operator:
In our experiments, we wantedc to figure out and confirmed the function of pTet-COMT. In other words, only we obtained the complete pure enzymes, we could find out its function. Thus, firstly we linked COMT 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 pTet-COMT was expressed successfully in E.coli BL21( DE3):
When the plasmid was constructed successfully, we must detect whether the enzyme we obtain can express in E.coli system. Therefore, we transferred the pTet- COMT plasmid into E.coli BL21 (DE3) strain [3] for expression. What’s more, in order to find out where the protein exactly existed, we used the SDS-PAGE to detect the different compositions of the bacteria lysate (Figure.2). In the figure2, line1-7 presented cell lysate,supernatant of cell lysate, precipitate of cell lysate, flow through, wash, elution, 10x concentrated elution, and positive control (purified COMT protein) respectively.And there existed a light target band in the line 7(Fig.6), and a obvious band with target size 40kDa in the line 1 and line 3 respectively. the SDS-PAGE result indicated that pSB1C3-pTet-COMT was expressed sucessfully in E,coli<i>, but most expressed protein were insoluble.
Prove the pTet-COMT really function in <i>E.coli system:
However, there was still a doubt that if the enzyme really works in the E.coli BL21 (DE3) system. Hence, to verify the pTet-COMT 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 pTet-COMT 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, Lee HY, Lee K, Back K (2014b) Caffeic acid O-methyltransferase is involved in the synthesis of melatonin by methylating Nacetylserotonin in Arabidopsis. J Pineal Res 57:219–227
[4] Byeon Y, Choi GH, Lee HY, Back K (2015a) Melatonin biosynthesis requires N-acetylserotonin methyltransferase activity of caffeic acid O-methyltransferase in rice. J Exp Bot 66:6917–6925
[5] Chen F, Tao Y, Jin C, Xu Y, Lin BX (2015) Enhanced production of polysialic acid by metabolic engineering of Escherichia coli. Appl Microbiol Biotechnol 99:2603–261
[6] Hardeland R (2015) Melatonin in plants and other phototrophs: advances and gaps concerning the diversity of functions. J Exp Bot 66:627– 646
[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] Lee HY, Byeon Y, Lee K, Lee HJ, Back K (2014b) Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localization. J Pineal Res 75:418– 426
//function/biosynthesis
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