35S promoter+translation initiation optimized sequence for dicot+Mass translation enhancer
The 35S promoter has a large area of application in genetically modified plants.The translation initiation optimized sequence for dicot is from the paper "translation initiation optimized sequence for dicot"(Nucleic Acids Research Volume 36, Issue 3Pp. 861-871 So Nakagawa1, Yoshihito Niimura2, Takashi Gojobori3,4, Hiroshi Tanaka1,2,* and Kin-ichiro Miura5)
The 35S promoter is a strong promoter derived from cauliflower mosaic virus. This constitutive promoter is widely used in transgenic plants to improve the level of the expression of foreign genes effectively."AAAAAAATG" was a kind of preferred nucleotide sequence used in dicot plants（ATG is the initiation codon）to enhance translation initiation.And insertion of ???GCT TCC TCC???(mass translation enhancer) after the initiator codon ATG can augment downstream gene-expression in plants.
We have to remove some restriction endonuclease cutting sites by changing a few bases.
translation initiation optimized sequence for dicot
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. These sequences were first discovered in the foot-and-mouth disease virus (FMDV).And since than many 2A-like sequences have been identified in other viruses and some parasites.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(from foot-and-mouth disease virus), P2A(from porcine teschovirus-1) and T2A(from Thosea asigna virus) to achieve our goal.And "GSG linker" is an oligopeptide of ???Gly-Ser-Gly??? between your protein and 2A peptide to enhance cleavage.
We have to use this part by synthesis from biological company because the virus is dangerous.
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.
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. These sequences were first discovered in the foot-and-mouth disease virus (FMDV).And since than many 2A-like sequences have been identified in other viruses and some parasites.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(from foot-and-mouth disease virus), P2A(from porcine teschovirus-1) and T2A(from Thosea asigna virus) to achieve our goal.And GSG linker is an oligopeptide of ???Gly-Ser-Gly??? between your protein and 2A peptide to enhance cleavage.
We have to use it by chemosynthesis because the virus is dangerous.
TA29 promoter is a tissue-specific(tapetal cells) promoter found in tobacco.
The GC1 promoter drives strong reporter expression in guard cells of Arabidopsis and tobacco plants. It provides a potent research tool for targeted guard cell expression.
Leading formate dehydrogenase into chloroplast.
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.
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 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).
AHA2 belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell, generating a proton gradient that drives the active transport of nutrients by proton symport.We want to enlarge the tobacco AHA2 to
The heat shock protein ( HSP ) terminator increases mRNA levels of both transiently and stably expressed transgenes in plants effectively.
It???s a kind of terminator derived from cauliflower mosaic virus.
It???s a quite common terminator in expression system of plants.
Conversion of the iGEM parts registry to SBOL2.1