Team:UESTC-China/BioBrick

From 2014.igem.org

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<h2>FALDH <a href="http://parts.igem.org/Part:BBa_K1537026"> (<u>BBa_K1537026</u>) </a></h2>
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<h2><i>FALDH</i> <a href="http://parts.igem.org/Part:BBa_K1537026"> (<u>BBa_K1537026</u>) </a></h2>
<p style="color:#1b1b1b;">
<p style="color:#1b1b1b;">
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The glutathione-dependent formaldehyde dehydrogenase (FALDH) plays a key role in formaldehyde metabolism. 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 combine 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. FALDH is identified as an enzyme expressed in the cytoplasm. If we make FALDH 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 combine 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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<h2>FDH <a href="http://parts.igem.org/Part:BBa_K1537025">(<u>BBa_K1537025</u>)</a></h2>
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<h2><i>FDH</i> <a href="http://parts.igem.org/Part:BBa_K1537025">(<u>BBa_K1537025</u>)</a></h2>
<p style="color:#1b1b1b;">
<p style="color:#1b1b1b;">
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Formate dehydrogenase (FDH) 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 was completed.
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Formate dehydrogenase (FDH) 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 <i>FDH</i> from arabidopsis thaliana was completed.
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<h2>HPS and PHI <a href="http://parts.igem.org/Part:BBa_K1537024"> (<u>BBa_K1537024</u>) </a></h2>
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<h2><i>HPS</i> and <i>PHI</i> <a href="http://parts.igem.org/Part:BBa_K1537024"> (<u>BBa_K1537024</u>) </a></h2>
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<p style="color:#1b1b1b;">
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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-Hexuloiso-merase (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.
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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-Hexuloiso-merase (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.
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  <h1 class="SectionTitles" style="width:1100px;">Part for safety</h1>
  <h1 class="SectionTitles" style="width:1100px;">Part for safety</h1>
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<h2>AdCP <a href="http://parts.igem.org/Part:BBa_K1537027"> (<u>BBa_K1537027</u>) </a></h2>
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<h2><i>AdCP</i> <a href="http://parts.igem.org/Part:BBa_K1537027"> (<u>BBa_K1537027</u>) </a></h2>
<p style="color:#1b1b1b;">
<p style="color:#1b1b1b;">
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Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Pawan Shukla has used a plant pathogen-induced gene, cysteine protease to induce 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>. Arachis diogoi 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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Considering the problem of environment and safety, we use male sterility system which prevents the horizontal transgene flow. Pawan Shukla has used a plant pathogen-induced gene, cysteine protease to induce 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>. Arachis diogoi cysteine protease  (<i>AdCP</i>) 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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<h2>AHA2 <a href="http://parts.igem.org/Part:BBa_K1537028"> (<u>BBa_K1537028</u>) </a></h2>
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<h2><i>AHA2</i> <a href="http://parts.igem.org/Part:BBa_K1537028"> (<u>BBa_K1537028</u>) </a></h2>
<p style="color:#1b1b1b;">
<p style="color:#1b1b1b;">
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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, showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening. Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plantgrowth. Therefore, in order to improve the ability of absorbing formaldehyde, we overexpresse 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, Yin Wang, et al, showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening. Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plantgrowth. Therefore, in order to improve the ability of absorbing formaldehyde, we overexpresse H+-ATPase (<i>AtAHA2</i>) in transgenic tobacco guard cells , resulting in a significant effect on light-induced stomatal opening.
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It’s chloroplast transit peptides. We use them to guide HPS-PHI and FALDH into chloroplast.
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They both are chloroplast transit peptides. We use them to guide HPS-PHI and FALDH into chloroplast.
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  <h1 class="SectionTitles" style="width:1100px;background-color: #2828FF">Improved Parts</h1>
  <h1 class="SectionTitles" style="width:1100px;background-color: #2828FF">Improved Parts</h1>
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<h2>35S promoter + translation initiation optimized sequence for dicot + mass translation enhancer<a href="http://parts.igem.org/Part:BBa_K1537015">(<u>BBa_K1537015</u>)</a></h2>
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<h2>35S promoter<a href="http://parts.igem.org/Part:BBa_K1537015">(<u>BBa_K1537015</u>)</a></h2>
<h2>Pre-existing Part:<a href="http://parts.igem.org/Part:BBa_K414002"><u>BBa_K414002</u></a></h2>
<h2>Pre-existing Part:<a href="http://parts.igem.org/Part:BBa_K414002"><u>BBa_K414002</u></a></h2>
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<p>We added translation initiation optimized sequence for dicot and mass translation enhancer after 35S promoter to enhance gene-expression in plants.<br/><br/><br/></p>
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<p>Our 35S promoter is based on BBa_K414002, and we also add translation initiation optimized sequence for dicot and MASS translation enhancer after 35S promoter to enhance gene-expression in plants.<br/><br/><br/></p>
<h2>GSG linker+P2A <a href="http://parts.igem.org/Part:BBa_K1537016">(<u>BBa_K1537016</u>)</a></h2>
<h2>GSG linker+P2A <a href="http://parts.igem.org/Part:BBa_K1537016">(<u>BBa_K1537016</u>)</a></h2>
<h2>GSG linker+T2A <a href="http://parts.igem.org/Part:BBa_K1537017">(<u>BBa_K1537017</u>)</a></h2>
<h2>GSG linker+T2A <a href="http://parts.igem.org/Part:BBa_K1537017">(<u>BBa_K1537017</u>)</a></h2>
<h2>Pre-existing Part: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1199016"><u>BBa_K1199016</u>, </a> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1199046"><u>BBa_K1199046</u></a></h2>  
<h2>Pre-existing Part: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1199016"><u>BBa_K1199016</u>, </a> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1199046"><u>BBa_K1199046</u></a></h2>  
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<p>We added GSG linker to enhance cleavage. In addition, we use 3 kinds of 2A rather than only one 2A in a vetor.<br/></p>
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<p>We added GSG linker to enhance cleavage. In addition, we use 3 kinds of 2As rather than only one 2A in a vector.<br/></p>
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GSG linker is an oligopeptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage.
GSG linker is an oligopeptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage.
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The 18~22 amino acids 2A self-cleaving oligopeptides can be used for co-expression of multiple, discrete proteins from a single ORF (Fig.1).Based on highly in efficient peptide bond formation between glycineand proline residues within the 2A peptide, placementof 2A peptide sequence as a linker region betweentandem cDNA’s allows the stoichiometric translation ofmultiple unfused protein products. To minimize therisk of homologous recombination, it is important to use different 2A peptide sequences if morethan two genes are being linked.The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammaliancells, yeast, and plants.In our project,we use F2A(from foot-and-mouth disease virus), P2A(from porcine teschovirus-1) and T2A(from Thosea asigna virus) to achieve our goal.
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The 18~22 amino acids 2A self-cleaving oligopeptides can be used for co-expression of multiple, discrete proteins from a single ORF. Based on highly inefficient peptide bond formation between glycine and proline residues within the 2A peptide, placement of 2A peptide sequence as a linker region between tandem cDNA’s allows the stoichiometric translation of multiple unfused protein products. To minimize the risk of homologous recombination, it is important to use different 2A peptide sequences if more than two genes are being linked. The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammalian cells, yeast, and plants. In our project, we use F2A, P2A and T2A to achieve our goal.
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Latest revision as of 03:10, 18 October 2014

UESTC-China