Team:Wisconsin-Madison/Parts

Basic Parts

• GSMT Gene
• SDMT Gene
• ProU Promoter
• MetK Gene
• NudF Gene
• PhotoSystem Promoter

Composite Parts

• ProU Promoter : RBS + RFP + TERM
• P

Basic Parts

GSMT is a plasmid gene (854 bp) that codes for the enzyme that catalyze the first reaction in the salt tolerant pathway that we are interested. GSMT stands for Glycine Sarcosine N-Methyl Transferase. It catalyzes the conversion from glycine to sarcosine, using SAM as a methyl donor. In our project, we have this GSMT synthesized using MR. Gene. Naturally, this gene can be found in Galdieria sulphuraria, an extremephile that establishes high resistance to osmotic stress as well as acidic, thermal stresses and toxic metals. We PCR amplified gene GSMT with iGEM designated cutting sites and ribosome binding sites and biobricked it with pSB1A2.

SDMT is a plasmid gene (900 bp) that codes for the enzyme that catalyze the first reaction in the salt tolerant pathway that we are interested. This enzyme of this pathway is SDMT, which stands for Sarcosine Dimethylglycine Methyl Transferase. It catalyzes two steps of methylation, both that of sarcosine and dimethylglycine, to our final product, betaine, again using SAM as the methyl donor. The gene we used came from Galdieria sulphuraria, an extremephile that establishes high resistance to osmotic stress as well as acidic, thermal stresses and toxic metals. In our project, we really appreciate that Center for Eukaryotic Structural Genomics at University of Wisconsin-Madison graciously gave us the plasmid containing this gene. We PCR amplified gene SDMT with iGEM designated cutting sites and ribosome binding sites and biobricked it with pSB1A2.

The ProU promoter (658 bp) was obtained from wild type K12 E. Coli Genomic DNA. Pro U controls transcription of the Pro U operon which contains three sub-units instrumental in bringing choline and proline into the cell. The promoter is most active during times of osmotic stress. We PCR amplified the gene proU with iGEM designated cutting sites and ribosome binding sites and biobricked it with pSB1A2.

metK is a plasmid gene (1200 bp) that codes for the enzyme that catalyzes the reaction which produces a methyl donor which is used in the conversion of Glycine to Glycine betaine. The enzyme for this reaction is S-adenosyl-methionine (SAM) synthetase. S-adenosyl-methionine produced from Methionine and one ATP molecule in the presence of SAM synthetase. Three SAM molecules are used in the methylation of Glycine to Glycine betaine (trimethyl glycine). In our project Glycine betaine (an osmoprotectant) was synthesized in E. coli to increase salt tolerance. It was hypothesized that an over expression of metK would lead to an increased production of Glycine betaine.

NudF is a plasmid gene (560 bp) that codes for the protein responsible for the enzymatic reaction in mevalonent pathway to form Dimethylallyl pyrophosphate (DMAPP). Specifically, in our project, we used it in the last step of mevalonate pathway to produce biofuels iso-pentenol. We PCR amplified gene nudF with iGEM designated cutting sites and ribosome binding sites and biobricked it with pSB1A2.

The psbA1 promoter (277 bp) was originally found in Synechococcus elongates cyanobacterium (PCC 7942). The promoter is one of the three psbA promoters that are responsible for regulating photosystem II protein transcription. Because the promoter is used to regulate photosystem II protein, the promoter activity is based off light conditions. In conditions of intense light (750 μE m-2 s-1), the promoter activity is decreased. The UW-Madison iGem team obtained the promoter gene by synthesizing the gene with biobrick cut sites.

Composite Parts

The ProU promoter (658 bp) was obtained from wild type K12 E. Coli Genomic DNA. Pro U controls transcription of the Pro U operon which contains three sub-units instrumental in bringing choline and proline into the cell. The promoter is most active during times of osmotic stress. Placing the proU promoter before the gene codes for red fluorescence protein with ribosomal binding site would enhance the level of red fluorescence expression under salt stress. We PCR amplified the gene proU with iGEM designated cutting sites and ribosome binding sites and biobricked it with pSB1A2.