Difference between revisions of "Part:BBa K1963007"

Latest revision as of 12:09, 23 October 2016

Device for rhamnose-dependent expression of an OsmY.

An assembled device that contains a rhamnose-responsive promoter, a strong RBS and a biobrick encoding the E. coli OsmY protein.

Usage and Biology

The E. coli OsmY protein is really interesting. Originally dubbed "osmotically-induced protein Y" it is synthesised with an N-terminal signal peptide (MTMTRLKISKTLLAVMLTSAVATGSAYA) most likely of the Sec type. While the protein has been reported to be periplasmic, it also appears to be one of the few fully secreted proteins that E. coli K-12 synthesises. This property has been used in SynBio projects (e.g. Steen et al. 2010) and in protein production projects (e.g. Le & Wang 2014).

An OsmY-encoding biobrick was assembled by Washington iGEM in 2012 (BBa_K892008) and this was potentially useful for secreting attached fusion proteins from an E. coli chassis.

OsmY has an cleaved N-terminal signal peptide, so it was not possible to add an epitope tag to the N-terminus. The original OsmY biobrick was assembled to the RFC[10] standard to contains double stop codons - and so not straightforward to slot in an epitope tag at the other end. PCR was therefore used to add a HA-tag sequence to OsmY. This allowed facile detection of the produced protein (Figure 1).

The E. coli host strain MG1655 was transformed with BBa_K1963007 and grown in liquid culture (5 ml) in the presence or absence of varying amounts of L-Rhamnose (Figure 1). A control transformation of the biobrick encoding just the rhamnose promoter was also carried out. Following overnight growth, cultures were separated into whole cell and culture supernatant fractions and analysed by SDS-PAGE and Western immunoblotting (Figure 1).

Figure 1: OsmY Production is Induced by Rhamnose and a Portion of the Protein is Secreted. Whole cell (A) and culture supernatant blots (B) of pRhamnose-OsmY-HA construct, depicted in (C) in the presence of increasing concentrations of Rhamnose. Anti-HA Western immunblot shows whole cell production and secretion of OsmY-HA in the presence of Rhamnose.

Sequence and Features