Uczestnistwo w konferencji - EcoFactory, a multipromoter explorer expression system for Escherichia coli.

Termin: 08-09-2015
Charakter: Krajowy

Omówienie stworzonych konstuktów genetycznych, zawierający superfolder HIS GFP, ulegający ekspresji pod kontrolą czterech induktorów- arabinozy, melibiozy, ramnozy i ksylozy. 

Minimal, independently regulated promoters of different expression levels are important tools for synthetic biology to build complex but compact genetic systems. It is important for crystallographers to produce recombinant proteins of high purity and for pharmaceutical industry to make the whole process free of toxic compounds. Ideally the expression level should be tuneable, induction - non-toxic, mRNA translation – free of mistakes, with perfect folding , recombinant protein – soluble and homogenous, and finally its purification - fast and reproducible. We prepared a eries of plasmids expressing his-tagged superfolder GFP (Tian ..) under the control of four nontoxic inducers: arabinose, melibiose, rhamnose and xylose. The four promoters are more controllable than the most popular lactose induced T7RNA polymerase dependent systems. The coding sequences are transcribed by the cellular RNA polymerase which is slower and more accurate than T7 RNA polymerase. SfGFP can be used to compare activities of standard, modified, and minimal promoters during growth on different carbon sources and as well as a probe of a fused protein solubility. We present a comprehensive comparison of the sfGFP expression driven by inducible promoters with the insulated, constitutive promoters proC and proD as standards (Davis 2009). Two pairs of primers should be sufficient to clone an ORF of interest as a protein fused to sfGFP or to replace it in all vectors of the multipromoter system.


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