Team:DTU Denmark/parts

This biobrick can be fused to any protein of interest and enables careful control of protein level by increasing degradation rate.

This destabilization sequence for rapid protein turnover consists on the C-terminal domain of Saccharomyces cerevisiae cyclin 2 (CLN2) gene. It has been shown that this region of the protein, which is rich in PEST motifs, leads to a destabilization of a protein. Hence this Tag can be used to increase the turn-over rate of a protein. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues changes the half-life of a GFP from 7h and down to 30 minutes.

Now the parts registry also offers a GFP for yeast - codon optimized and enhanced. By expanding the host organism, this is an improvement of an existing biobrick: BBa_E0040

This constitutively expressed GFP, originally from Aequorea victoria was codon optimized and developed as a reporter for gene expression in Saccharomyces cerevisiae according to Cormak et al (1997). In addition, two amino acid changes were included enabling a far more bright florescence compared to the wild type in E.coli. Maximum absorbance: 490 nm.

A fast degradable GFP that has a half-life of 30 min. This variant of biobrick BBa_K194001 has been destabilized by fusing it with biobrick BBa_K194000. In the article by Mateus and Avery: "Destabilized green Fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry" they show that addition of these 178 carboxyl-terminal amino acid residues of the Cln2 PEST signal, changes the half-life of a GFP from 7h down to 30 minutes.

This biobrick will allow insertion of PCR fragments using the novel USERTM biobrick assembly standard (BBF RFC 39). It contains a PacI/Nt.BbvCI USER cassette for insertion of PCR fragments using the USER cloning standard. PCR fragments containing uracil is treated with a uracil DNA glycosylase that removes the uracil exposing a predesigned 8bp overhang allowing for cloning without the need for ligase