Difference between revisions of "Part:BBa K1680016"

Revision as of 01:35, 26 September 2015

pRS316 yeast shuttle vector with Biobrick MCS

This part is a biobricked shuttle vectors for Saccharomyces cerevisiae. It is part of a vector series with different auxotrophy markers (Part:BBa_K1680014, Part:BBa_K1680015). The vector was made compatible with the RFC10 standard and also contains a RFC10 cloning site. This part is derived from the wild type pRS316 vector Part:BBa_K950011.

This part encodes for an ampicillin resistance gene and replicates to high-copy numbers in E. coli. Furthermore, the CEN6/ARS4 (Chromosome VI centromere/Autonomously Replicating Sequence 4) cassette ensures that the plasmid stays at a low copy number in yeast, because they are treated as pseudo chromosomes. The vector also encodes for a URA3 gene, which is used as an auxotrophy marker in yeast.

This part is mutagenized version of the wild type pRS316 vector (Sikorski 1989).

A PstI and SacI restriction site in the backbone were removed by mutagenesis PCR (see lane 1-3 in figure). Exchange of the /wild type MCS for RFC10 compatible MCS could be shown by restriction with SalI restriction: The enzyme linearizes the vector with old MCS (lane 4) but does not cut the vector with the new MCS, as indicated by presence of supercoiled DNA (lane 5).

Agarose Gel of different Versions of the pRS313 vector. Lane 1: wild type vector, cut with PstI; lane 2: mutagenised vector (PstI site) cut with PstI; lane 2: mutagenised vector (SacI site) cut with SacI; lane 3: mutagenised vector cut with SalI; lane 4: vector with RFC10 MCS cut with SalI.

Sequence and Features

This part is a RFC10 compatible shuttle vector for yeast.