PtoMYB46 reduces the expression of PtoMATE directly or indirectly through PMAT, thereby reducing the secretion of citric acid (CA) and ultimately promoting Pb2+ uptake. Meanwhile, PtoMYB46 targets auxin response factor 2 (ARF2) and reduces its expression, thus positively regulating plant growth.

In order to carry forward the spirit of iGEM, and inherit and spread the value of iGEM, our team uses directed evolution to screen positive selection sites and optimize the function of target genes by means of bioinformatics. At the same time, the vector itself is optimized and ribosome binding sites are added to improve the translation efficiency of the protein. Through the transformation of these part, our goal is to achieve efficient absorption of lead ions in deep soil.

Heavy-metal pollution is a growing environmental problem around the world. We specially studied and investigated environmental protection. Lead (Pb2+) is one of the most toxic heavy-metal contaminants has harmed agroforestry production, and also the health of humanity. In China, there are 20 million hectares of land which had suffered from heavy-metal pollution. It can lead to climate change and a series of severe consequences. Grain pollution is estimated to reach as high as 12 million tons per year, indicating that plants are robbed of nutrients, moreover, the toxicity would endanger the human race. The heavy-metals pollution has threatened the quality of human life profoundly. For the reasons mentioned above, we expect to accomplish effective absorption of lead (Pb2+) ions in deep soil using synthetic biology and try our best to improve the situation that's been going around on Earth right now. PtoMYB46 reduces the expression of PtoMATE directly or indirectly through PMAT, thereby reducing the secretion of citric acid (CA) and ultimately promoting Pb2+ uptake. Meanwhile, PtoMYB46 targets auxin response factor 2 (ARF2) and reduces its expression, thus positively regulating plant growth. We are committed to applying synthetic biology to the problem of heavy-metal (HM) pollution in soil. By the means of synthetic biology, our team has engineered genes into plants that can absorb residual metals in the soil.

We Screened positive loci by directed evolution to optimize gene structure. Positive selected amino acid sites of PtoMYB46: Phe 18 Gln, Met 35 Tyr, Leu 52 Ser. inserted it into the BamHI site of pBI121 vector. At the same time, the vector itself is optimized and ribosome binding sites are added to improve the translation efficiency of the protein. The certificate of recombinant plasmid sequencing results is as Figure1.

We Screened positive loci by directed evolution to optimize gene structure. Positive selected amino acid sites of PtoMYB46: Phe 18 Gln, Met 35 Tyr, Leu 52 Ser. inserted it into the KpnI site of pYES2 vector. At the same time, the vector itself is optimized and ribosome binding sites are added to improve the translation efficiency of the protein. The certificate of recombinant plasmid sequencing results is as Figure2.

We also created new parts to make contribution to future iGEM teams.

PtoMYB46 reduces the expression of PtoMATE directly or indirectly through PMAT, thereby reducing the secretion of citric acid (CA) and ultimately promoting Pb2+ uptake. Meanwhile, PtoMYB46 targets auxin response factor 2 (ARF2) and reduces its expression, thus positively regulating plant growth.

A Ribosome Binding Site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation. Translation initiation in bacteria almost always requires both an RBS sequence and a start codon.

pBI121-PtoMYB46 is a composite part that could be used for PtoMYB46 protein Overexpression.

pBI121-PtoMYB46 is a composite part that could be used for PtoMYB46 protein Overexpression.

pBI121 vector is a binary Agrobacterium tumefaciens expression vector, which can be efficiently transfected into plants. The vector is derived from pB221 and Bin19 vectors. The vector contains ori replication elements from ColE1, pROK1 elements from CaMV, Escherichia coli neomycin phosphotransferase II gene (Neomycin phosphotransferase II Gene, NptII), neomycin resistance gene (Neomycin resistant gene, Neo), sensitive gene Beta glucuronidase (glucuronidase), Ter Agrobacterium tumefaciens Ti plasmid carmine synthetase. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Kana+ resistance can be used to screen the right colony and can insert target genes in MCS. This plasmid backbone can be used to express different proteins in the future.

Our team has constructed the PtoMYB46 gene overexpression plasmids, both the DNA fragments and the vector were digested by double enzymes, verified by agarose gel electrophoresis, and the correct recombinant plasmid was extracted and transformed into GV3101.

The pYES2 vector is designed for native expression of protein of interest in S. cerevisiae. CYC1 Terminator can effectively terminate the transcription of mRNA.

pYES2-PtoMYB46 is a composite part that could be used for PtoMYB46 protein expression in yeast cells (BY4741). pYES2 is a 5.9 kb vector designed to induce the expression of recombinant protein in Saccharomyces cerevisiae (Saccharomyces cerevisiae). Yeast GAL1 promoter can be induced to express PtoMYB46 in Saccharomyces cerevisiae by high level of galactose. Our team has constructed the PtoMYB46 protein expression plasmids, both the DNA fragments and the vector were digested by double enzymes, verified by agarose gel electrophoresis, and the correct recombinant plasmid was extracted and transformed into BY4741.

PtoMYB46 reduces the expression of PtoMATE directly or indirectly through PMAT, thereby reducing the secretion of citric acid (CA) and ultimately promoting Pb2+ uptake. Meanwhile, PtoMYB46 targets auxin response factor 2 (ARF2) and reduces its expression, thus positively regulating plant growth. positive selected amino acid sites of PtoMYB46: Phe 18 Gln, Met 35 Tyr, Leu 52 Ser.

pBI121-PtoMYB46-1 is a composite part that could be used for PtoMYB46 protein Overexpression.

pBI121 vector is a binary Agrobacterium tumefaciens expression vector, which can be efficiently transfected into plants. The vector is derived from pB221 and Bin19 vectors. The vector contains ori replication elements from ColE1, pROK1 elements from CaMV, Escherichia coli neomycin phosphotransferase II gene (Neomycin phosphotransferase II Gene, NptII), neomycin resistance gene (Neomycin resistant gene, Neo), sensitive gene Beta glucuronidase (glucuronidase), Ter Agrobacterium tumefaciens Ti plasmid carmine synthetase. The vector is a high-copy-number plasmid. When expressed in the prokaryotic system, the Kana+ resistance can be used to screen the right colony and can insert target genes in MCS. This plasmid backbone can be used to express different proteins in the future. PtoMYB46 reduces the expression of PtoMATE directly or indirectly through PMAT, thereby reducing the secretion of citric acid (CA) and ultimately promoting Pb2+ uptake. We Screened positive loci by directed evolution to optimize gene structure. Positive selected amino acid sites of PtoMYB46: Phe 18 Gln, Met 35 Tyr, Leu 52 Ser. inserted it into the BamHI site of pBI121 vector. At the same time, the vector itself is optimized and ribosome binding sites are added to improve the translation efficiency of the protein.

pYES2-PtoMYB46 is a composite part that could be used for PtoMYB46 protein expression in yeast cells (BY4741). pYES2 is a 5.9 kb vector designed to induce the expression of recombinant protein in Saccharomyces cerevisiae (Saccharomyces cerevisiae). Yeast GAL1 promoter can be induced to express PtoMYB46 in Saccharomyces cerevisiae by high level of galactose. Our team has constructed the PtoMYB46 protein expression plasmids, both the DNA fragments and the vector were digested by double enzymes, verified by agarose gel electrophoresis, and the correct recombinant plasmid was extracted and transformed into BY4741. We Screened positive loci by directed evolution to optimize gene structure. Positive selected amino acid sites of PtoMYB46: Phe 18 Gln, Met 35 Tyr, Leu 52 Ser. inserted it into the KpnI site of pYES2 vector. At the same time, the vector itself is optimized and ribosome binding sites are added to improve the translation efficiency of the protein.