pET24a(+)_mRuby2
$30.00
From Kyle J. Lauersen, Jan H. Mussgnug, Olaf Kruse, Center for Biotechnology (CeBiTec), Bielefeld University, Germany, September 2014
NCBI accession number: KM504961
The pOpt system was originally built with four fluorescent protein standards and one luciferase.
The Gaussia princeps luciferase is available as a secreted product standard from Avidity: https://www.avidity.com.
However, the four fluorescent reporters, mCerulean3 (a bright cyan FP variant), mVenus (a bright yellow FP variant), Clover (a very bright green FP variant), and mRuby2 (a bright red FP variant from Entacmaea quadricolor), are not commercially available as protein standards.
Since the pOptimized expression system contains two introns (1 and 2 of RBCS2), it would require specialized reconstruction of the genes without introns for the researcher to make Escherichia coli expression constructs to use as standards.
We, therefore, created E. coli codon optimized versions of the four fluorescent reporters containing the extra amino acids on C- and N-termini that are a product of the pOpt restriction sites, and included the StrepII tag on their C-terminus as is also found in the pOpt reporters. The protein products from these E. coli expression cassettes are identical to those produced from their pOpt citosolic localized counterparts.
The E. coli expression constructs are housed in the pET24a(+) backbone, driven by the T7 promoter, and are therefore inducible by addition of IPTG.
pET24a(+) is Kanamycin resistant.
The cell lines are E. coli KRX, which are designed for high recombinant protein expression purposes.
These cell lines and plasmids are usable directly without any additional cloning in standard E. coli fermentations and StrepII affinity chromatography.
Lauersen KJ, Kruse O, Mussgnug JH (2015) Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Appl Microbiol Biotechnol. Jan 15 Lumbreras V, Purton S (1998) Recent advances in chlamydomonas transgenics. Protist 149:23-7 Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153:401-12 Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 77:221-9