Difference between revisions of "Part:BBa K1963007"
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The <i>E. coli</i> 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 <i>E. coli</i> K-12 synthesises. This property has been used in SynBio projects (e.g. Steen <i>et al.</i> 2010) and in protein production projects (e.g. Le & Wang 2014). | The <i>E. coli</i> 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 <i>E. coli</i> K-12 synthesises. This property has been used in SynBio projects (e.g. Steen <i>et al.</i> 2010) and in protein production projects (e.g. Le & Wang 2014). | ||
| − | An OsmY-encoding biobrick was assembled by Washington iGEM in 2012 ( | + | An OsmY-encoding biobrick was assembled by Washington iGEM in 2012 (<partinfo>BBa_K892008</partinfo>) and this was potentially useful for secreting attached fusion proteins from an <i>E. coli</i> 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). | 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). | ||
Revision as of 10:17, 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).
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]