Team:UESTC-China/Design

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<h1 style="color:#1b1b1b; position:relative; left:0px; padding:15 5px; font-size:40px; font-family: calibri, arial, helvetica, sans-serif; font-weight: bold;font-style: Italic; text-align:center; width:1140px;">Design</h1>
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<p style="color:#1b1b1b;">In this project, our objective is to further increase plant formaldehyde uptake and metabolism ability using synthetic biology methods. We find some genes encoding key enzymes related to formaldehyde metabolic pathways from microorganism. They are 3-hexulose-6-phosphate (HPS), 6-phospho-3-hexuloisomerase (PHI), formaldehyde dehydrogenase (FALDH) and formate-dehydrogenase (FDH). These genes are inserted into plants and will work to promote formaldehyde metabolism. For security reasons, we also add gene AdCP into our project because of its capability to lead to pollen abortion. At the same time, chloroplast transformation is taken into consideration to decrease the probability of gene flow.
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<p style="color:#1b1b1b;">In order to further increase the plant ability of formaldehyde uptake and metabolism by synthetic biology technology, we choosed four enzyme-coding genes related to formaldehyde metabolic pathways from microorganism and plant: 3-hexulose-6-phosphate (HPS), 6-phospho-3-hexuloisomerase (PHI), formaldehyde dehydrogenase (FALDH) and formate-dehydrogenase (FDH). These genes are transformed into plants and will promote formaldehyde metabolism. For security reasons, we also induce <i>AdCP</i> gene into our plans because of its capability to lead to pollen abortion. At the same time, chloroplast transformation is taken into consideration to avoid gene flow and improve gene expression.
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The ribulose monophosphate (RuMP) pathway is one of the HCHO-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compounds as the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes HCHO to D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose 6-phosphate (Hu6P), and 6-phospho-3- hexuloisomerase (PHI), which converts Hu6P to fructose 6-phosphate (F6P).The two key enzymes work in chloroplast both.We will use fusion expression to conduct heterologous expression in tobacco <i>( Li-mei Chen et al,2010)</i>. Here are some datas from the paper.  
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The ribulose monophosphate (RuMP) pathway is one of the formaldehyde-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compounds as the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes formaldehyde to D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose-6-phosphate (Hu6P), and 6-phospho-3-hexuloisomerase (PHI), which converts Hu6P to fructose 6-phosphate (F6P). The two key enzymes work in chloroplast both. We will use fusion expression method to conduct heterologous expression in tobacco (<i>Chen et al., 2010</i>).  
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<strong>Fig.1</strong>
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Schematic Representation of the Bacterial RuMP Pathway and the Plant Calvin-Benson Cycle. HPS and PHI denote 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase respectively. The abbreviations for several sugar phosphates are as follows: Ru5P, ribulose 5-phosphate; Hu6P, hexulose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; RuBP, ribulose1, 5-bisphosphate; 3-PGA, 3-phosphoglyce-rate. The other metabolites in the pathway are symbolized merely by their carbon numbers for simplicity <i>(Li-mei Chen et al,2010)</i>.  
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Schematic Representation of the Bacterial RuMP Pathway and the Plant Calvin-Benson Cycle. HPS and PHI denote 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase respectively. The abbreviations for several sugar phosphates are as follows: Ru5P, ribulose 5-phosphate; Hu6P, hexulose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; RuBP, ribulose1, 5-bisphosphate; 3-PGA, 3-phosphoglyce-rate. The other metabolites in the pathway are symbolized merely by their carbon numbers for simplicity <i></i>.  
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The glutathione-dependent formaldehyde dehydrogenase (FALDH) plays a key role in formaldehyde metabolism (Fig.3). FALDH is identified as an enzyme expressed in the cytoplasm. If we make FALDH over-express in plants, we can enhance plants’ tolerance to HCHO and increase the ability of plants to absorb HCHO. In the process of metabolism of formaldehyde, the formaldehyde may first combined with glutathione (GSH) to form the product of S-hydroxymethyl glutathione (HM-GSH), then FALDH in cytoplasm will catalyzes the formation of a S-formyl glutathione (F-GSH). Next the F-GSH will be hydrolyzed to formate (HCOOH) and GSH by S-formyl glutathione hydrolase (FGH).
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The glutathione-dependent formaldehyde dehydrogenase (FALDH) plays a key role in formaldehyde metabolism (Fig.3). FALDH is identified as an enzyme expressed in the cytoplasm. If we make <i>FALDH</i> over-express in plants, we can enhance plants’ tolerance to formaldehyde and increase the ability of plants to absorb formaldehyde. In the process of metabolism of formaldehyde, the formaldehyde may first combined with glutathione (GSH) to form the product of S-hydroxymethyl glutathione (HM-GSH), then FALDH in cytoplasm will catalyzes the formation of a S-formyl glutathione (F-GSH). Next the F-GSH will be hydrolyzed to formate (HCOOH) and GSH by S-formyl glutathione hydrolase (FGH).
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<strong>Fig.2</strong>  a. <img align="top" src="https://static.igem.org/mediawiki/2014/0/04/C13-nmr.gif"> spectra from leaf extracts of transgenic tobacco plant treated with gaseous <img align="top" src="https://static.igem.org/mediawiki/2014/e/e7/H13CHO.gif"> for 2 h. b. <img align="top" src="https://static.igem.org/mediawiki/2014/0/04/C13-nmr.gif"> spectra from leaf extracts of WT treated with gaseous <img align="top" src="https://static.igem.org/mediawiki/2014/e/e7/H13CHO.gif"> for 2 h. c. The extract from WT plant leaves without <img align="top" src="https://static.igem.org/mediawiki/2014/e/e7/H13CHO.gif"> treatment was used to monitor the background <img align="top" src="https://static.igem.org/mediawiki/2014/0/04/C13-nmr.gif"> signal levels <i>(H Nian et al,2013)</i>.  
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<strong>Fig.2</strong>  a. <img align="top" src="https://static.igem.org/mediawiki/2014/0/04/C13-nmr.gif"> spectra from leaf extracts of transgenic tobacco plant treated with gaseous <img align="top" src="https://static.igem.org/mediawiki/2014/e/e7/H13CHO.gif"> for 2 h. b. <img align="top" src="https://static.igem.org/mediawiki/2014/0/04/C13-nmr.gif"> spectra from leaf extracts of WT treated with gaseous <img align="top" src="https://static.igem.org/mediawiki/2014/e/e7/H13CHO.gif"> for 2 h. c. The extract from WT plant leaves without <img align="top" src="https://static.igem.org/mediawiki/2014/e/e7/H13CHO.gif"> treatment was used to monitor the background <img align="top" src="https://static.igem.org/mediawiki/2014/0/04/C13-nmr.gif"> signal levels <i>(Nian et al., 2013)</i>.  
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Formate dehydrogenase is a mitochondrial-localized NAD-requiring enzyme while the HCOOH is getting into the mitochondrial,FDH will oxidize the formic acid into CO2, and reduce NAD+ to NADH with a high degree of specificity.In our project, the heterologous expression of FDH from arabidopsis thaliana in tobacco was completed.
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Formate dehydrogenase is a mitochondrial-localized NAD-requiring enzyme while the HCOOH is getting into the mitochondrial, FDH will oxidize the formic acid into CO2, and reduce NAD+ to NADH with a high degree of specificity. In our project, the heterologous expression of Arabidopsis <i>FDH</i> gene in tobacco was completed.
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<strong>Fig.3</strong> The abbreviations are as follows: FALDH:glutathione-dependent formaldehyde dehydrogena-se; FDH: Formate dehydrogenase; HM-GSH: S-Hydroxymethyl glutathione; Forml-GSH: Formyl glutathione; SMM cycle: Methionine cycle.
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<strong>Fig.3</strong> Folate-independent pathway. The abbreviations are as follows: FALDH:glutathione-dependent formaldehyde dehydrogenase; FDH: Formate dehydrogenase; HM-GSH: S-Hydroxymethyl glutathione; Forml-GSH: Formyl glutathione; SMM cycle: Methionine cycle.
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Stomata are microscopic pores surrounded by two guard cells and play an important role in the uptake of CO2 for photosynthesis. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: blue light receptor phototropin, plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. However, Yin Wang, et al (2014) showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening (Fig. 4). Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plant growth. Therefore,in order to strengthen the ability of absorbing formaldehyde, we overexpressed H+-ATPase (AtAHA2) in transgenic tobacco guard cells,resulting in a significant effect on light-induced stomatal opening.
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Stomata are microscopic pores surrounded by two guard cells and play an important role in the uptake of CO2 for photosynthesis. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: blue light receptor phototropin, plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. However, Wang et al (2014) showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening (Fig. 4). Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plant growth. Therefore, in order to strengthen the ability of absorbing formaldehyde, we overexpressed H+-ATPase (<i>AtAHA2</i>) in transgenic tobacco guard cells, resulting in a significant effect on light-induced stomatal opening.
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<strong>Fig.4</strong>  Typical stomata in the epidermis illuminated with light for 30 min (<i>Yin Wang,et al.2014</i>).
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<strong>Fig.4</strong>  Typical stomata in the epidermis illuminated with light for 30 min (<i>Wang et al., 2014</i>).
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In order to promise biology safety, we use male sterility systems which can be used as a biological safety containment to prevent horizontal transgene flow. Pawan Shukla et al (2014) has used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachisdiogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsispersonata.Arachisdiogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion.
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In order to promise biology safety, we use male sterility systems which can be used as a biological safety containment to prevent horizontal transgene flow. Shukla et al (2014) has used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, <i>Arachis diogoi</i> differentially expressed when it was challenged with the late leaf spot pathogen, <i>Phaeoisariopsis personata</i>. <i>Arachis diogoi</i> cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of <i>AdCP</i> transgenic plants showed ablated tapetum and complete pollen abortion.
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<strong>Fig.5</strong> Pollen germination of untransformed control plant and sterile transgenic plantsin vitro. Pollen grains were germinated on sucrose-boric acid medium and over 500 pollen grains were observed. a. Untansformed control plant pollen, b. Sterile pollen.Scale bar 25 μm (<i>Pawan Shukla et al 2014</i>).  
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<strong>Fig.5</strong> Pollen germination of untransformed control plant and sterile transgenic plants <i>in vitro</i>. Pollen grains were germinated on sucrose-boric acid medium and over 500 pollen grains were observed. a. Untansformed control plant pollen, b. Sterile pollen. Scale bar 25 μm (<i>Shukla et al., 2014</i>).  
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The production of HPS, PHI, and FDH are located in chloroplast, while the production of FALDH are located in cytoplasm. We used chloroplast transit peptides to locate these productions of genes. So we constructed different vectors with and without transit peptide. We hope to compare the ability of metabolizing HCHO of transgenic tobacco between different transgenic lines. We planned to constructed 11 vectors (Fig. 6), including two backbones, six mono-gene expression vectors and three multi-gene expression vectors (Fig. 7). piGEM003, piGEM004 and piGEM005 are individual mono-gene expression vectors with transit peptides, while piGEM006, piGEM006, piGEM008 are individual multi-gene expression vectors without transit peptides. piGEM009 is a multi-gene expression vector without any transit peptides, while piGEM011 is a multi-gene expression vector with three peptides. piGEM010 is a multi-gene expression vector with two transit peptides.
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The product of <i>HPS</i>, <i>PHI</i>, and <i>FDH</i> are located in chloroplast, while the product of <i>FALDH</i> are located in cytoplasm. We used chloroplast transit peptides to locate these products of genes. So we constructed different vectors with and without transit peptide. We hope to compare the ability of metabolizing formaldehyde of transgenic tobacco between different transgenic lines. We planned to constructed 11 vectors (Fig. 6), including two backbones, six mono-gene expression vectors and three multi-gene expression vectors (Fig. 7). piGEM003, piGEM004 and piGEM005 are individual mono-gene expression vectors with transit peptides, while piGEM006, piGEM006, piGEM008 are individual multi-gene expression vectors without transit peptides. piGEM009 is a multi-gene expression vector without any transit peptides, while piGEM011 is a multi-gene expression vector with three peptides. piGEM010 is a multi-gene expression vector with two transit peptides.
   
   
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<strong>Fig.6</strong>  The procedure we constructed our vectors.  
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<strong>Fig.6</strong>  The procedure of vectors construction.  
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<strong>Fig.7</strong> Schematic of vectors we constructed
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<strong>Fig.7</strong> Schematic of vectors construction.
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Latest revision as of 02:27, 18 October 2014

UESTC-China