Team:GDSYZX/safety
Introduction
When conducting an experiment or developing a new product, our first cosideration is always the biosafety. It indicates whether the product may be at risk in the experimental design, process and subsequent development. Our team's research is to transfer chlorogenic acid (CGA) into chassis which is rice through Hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase（HQT） regulation, so as to improve the content of chlorogenic acid in rice and then develop the healthy food that can help improve immunity. Therefore, we consulted many infenrences to evaluate the safety of CGA, HQT and rice to ensure the overall safety of our project. At the same time, in the course of experiments and human practice, we have taken various measures to ensure that all actions are based on safety.
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In The Project
CGA
Lonicera japonica Thunb., native to eastern Asia, blooms white and yellow flowers with sweet scents in the summer. The dried flower bud is named Flos Lonicerae Japonicae (FLJ), which is commonly used as an antipneumonia, antivirus, antioxidant, and antitumor agent and to enhance liver bile protection. FLJ is also widely used as a food additive in beverages and healthy teas. Previous studies have shown that FLJ has abundant polyphenols with high antioxidant activities. Among these polyphenols, CGA is considered the major bioactive component because of its numerous bioactive functions and its ability to improve the body’s own defenses. Chlorogenic acid is widely distributed in plants. Furthermore, it is one of the main polyphenols in the human diet. After years of testing, chlorogenic acid is a common active ingredient in Traditional Chinese medicine. Therefore, it has a wide range of health-promoting properties and has been widely studied. Many drugs that contain chlorogenic acid are approved by the Food and Drug Administration(FDA). At the same time, we did not find any references that mentioned chlorogenic acid was harmful to humans. Thus, chlorogenic acid is safe.
Chlorogenic acid biosynthetic pathway
Quinic acid and Caffeoyl Coenzyme A are precursors of chlorogenic acid in rice[2]，and rice contains three possible pathways for the production of chlorogenic acid: formed by the condensation of quinic acid and caffeoyl-CoA, formed by the reaction of quinic acid and caffeoyl glucose, and produced by the hydroxylation of coumaroylquinic acid[3].
We select the first pathway that HQT catalyze Caffeyol-Coa and quinic acid to synthesize the CGA. It can be seen from the reaction process that the synthesis of CGA catalyzed by HQT is safe. [4] The reaction pathway exists in rice, and we only enhance the expression of HQTgene in rice. Thus, our work will not produce substances that are not found in food crops and is safe.
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Environment
According to research, elevated levels of CGAs in transgenic tomato plants have been shown to give increased protection from harmful UV light [5] and enhanced microbial resistance [6]. More recently, it has been shown that the CGAs can act as pest resistance factors in ornamental plants [7]. At the same time, CGA does no harm to untargeted insects,because chlorogenic acid has been present in nature for many years,and these insects have evolved over thousands of years to provide a defense against these natural pesticides.Therefore, the overexpression of chlorogenic acid in rice seeds will not pose a threat to environmental security.Our project is based on protoplasts, which do not grow into intact plants, so the modified cells cannot survive in the natural environment.And we will obey the rules of iGEM, do not put engineering cells into the environment and inactivate them before we discard the engineering cells. The promoter GluD-1 used has the property of spatiotemporal expression, which is expressed only in seeds and not in leaves. Therefore, our recombinant vectors would not be expressed in leaf protoplasts and are not easy to contaminate other materials and tissues.
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Laboratory
Before entering the laboratory, our team conducted a course on life science laboratory safety and the use of conventional instruments.The course explains the current situation of laboratory safety, types of laboratory accidents and preventive measures, as well as the principle and operation process of laboratory instruments.
Before using the apparatus in the experiment, necessary disinfection should be done.Put on sterilized protective clothing, put on disposable gloves, spray your hands with alcohol, and wait for the alcohol to dry before starting the experiment. During the experiment, the laboratory guidelines should be strictly observed under the guidance of the tutors. At least two people should participate in the experiment at the same time to avoid accidental vocalization.
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Human Practices
The Lecture
We conducted activities in local low-risk areas and strictly adhered to local anti-epidemic policies. We publicized our program in the community and made sure it was in open areas and everyone presented wore a mask or using other protective measures. At the same time, we used the necessary protection measures such as sterilizing hands with alcohol and standing away from each other for vulnerable and vulnerable groups. This could also ensure the safety of our activities during the epidemic.
Questionnaire
The questionnaire related to the project was published on the Internet to investigate the public's awareness of honeysuckle and GENETICALLY modified food and to collect the social identification of our project. This survey was conducted entirely on the Internet, and a team member summarized the result of the questionnaire uniformly. With the meaures above, our HP successfully collected a large number of questionnaires and fulfilled the required social distance under the influence of Coronavirus safely.
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Reference
[1] Kundu A., Vadassery J. Chlorogenic acid-mediated chemical defence of plants against insect herbivores. Plant Biol. 2019;21(2):185–189.
[2] Huayan Zhao, Qingxi Shen, Shiyou Lu, Analysis of expression characteristics of rice caffeoyl-coenzyme A-O-methyltransferase gene (CCoAOMT), Science Bulletin, 2004.
[3] Ulbrich B，Zenk MH． Partial purification and properties of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase from higher plants［J］ ． Phytochem， 1979， 18:929.
[4] Cloning and characterization of a cDNA coding a hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase involved in chlorogenic acid biosynthesis in Lonicera japonica.Peng XX，Li WD，Wang WQ， et al． Planta Med，2010， 76: 1921.
[5]A Structural Basis for the Biosynthesis of the Major Chlorogenic Acids Found in C offee Plant Physiol. 2012 Sep; 160(1): 249–260.Published online 2012 Jul 20. doi: 10.1104/pp.112.202051
[6] Clé C, Hill LM, Niggeweg R, Martin CR, Guisez Y, Prinsen E, Jansen MA. (2008) Modulation of chlorogenic acid biosynthesis in Solanum lycopersicum: consequences for phenolic accumulation and UV-tolerance. Phytochemistry 69: 2149–2156
[7] Leiss KA, Maltese F, Choi YH, Verpoorte R, Klinkhamer PG. (2009) Identification of chlorogenic acid as a resistance factor for thrips in Chrysanthemum. Plant Physiol 150: 1567–1575
[8] Niggeweg R, Michael AJ, Martin C. (2004) Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol 22: 746–754