Parts

2014-PDX-1

PDX-1 is a transcriptional factor that activates insulin, somatostatin, glucokinase, islet amyloid polypeptide and glucose transporter type 2 gene transcription. It’s essential for development of pancreas and maintain of the hormone-producing phenotype of the beta-cell. PDX-1 is used here for glucose-dependent regulation of insulin gene expression.

PDX-1

PDX-1 is a transcription factor involved in the expression of insulin and insulin-related genes. Co-transfection of PDX-1 and MafA in mammalian cells induces cell-type conversion to insulin producing beta-cells.

2014-insulin-forget

The insulin gene consists three exons, which encode B chain, C peptide and A chain. We modified the protease recognition sites of insulin gene to make sure that the insulin gene product undergoes accurate modification. The signal peptide and C peptide are removed after transcription and translation and two disulfide bonds form between A chain and B chain, undergoing folding to become functional insulin.

Insulin (modified protease recognition sites)

Encodes the human insulin gene (full length). Two 6 bp sequences were inserted into two separate sites within the insulin gene to allow enzymatic digestion by the protease furin. Originally, proinsulin is cleaved three times with three different enzymes, prohormone convertases (PC1 and PC2), and carboxypeptidase E before being secreted as mature insulin. Since PC1 and PC2 expression is tissue specific, we replaced their recognition sites with that of furin, which is universally expressed, thus allowing this insulin gene to be expressed and processed in any somatic cell type.

2014-HXT1+CMV-forget

Part HXT1+CMV is used as promoter for target gene under the regulation of glucose, consisting of HXT-1 promoter and CMV promoter. HXT-1 is a low-affinity glucose transporter from Saccharomyces cerevisiae. There is a glucose response element in HXT-1 gene sensitive to glucose in human fibroblasts, which is used here as a glucose-dependent regulator.

2014-HXT1+EGFP+ADH1-forget

HXT-1+EGFP+ADH1 consists of HXT-1+CMV promoter, EGFP and terminator ADH1. HXT-1+CMV promoter controls EGFP expression under the regulation of glucose. EGFP is used here for testing of HXT1+CMV promoter and will be replaced by insulin gene.

HXT1-CMVmini-EGFP-tADH1

This is a composite part containing a glucose-sensitive promoter (BBa_K1328000) and a EGFP reporter with a tADH1 transcription terminator.
S. cerevisiae cells transformed with this part was cultured in glucose-free medium with glycerol as carbon-source for 8 hrs. They were then cultured for an additional hour with varying glucose levels (0, 5, 15, 25 mmol/L). A increasing level of GFP expression was observed with increasing levels of glucose concentration, in both microscopy and flow cytometry observations.