Part:BBa_K346033:Design
T7p(BBa_I719005)+RBS(B0034)+DsbA+MBP(periplasm display of mercury binding peptide)
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 529
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 529
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 317
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 529
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 529
Illegal AgeI site found at 149 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Maltose Binding Protein and DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, were selected as the candidates for the periplasmic fusion MBPs. According to previous work, Maltose Binging Protein, a SecB dependent protein encoded by the malE gene, conducts the posttranslational export to the periplasmic space.(NOTE: SecB is a chaperone which prevents the large nascent polypeptide from folding into a conformation that would interfere with translocation ).On the other hand, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design.
Source
.plasmid from Summers
References
Pazirandeh, M., L. A. Chrisey, J. M. Mauro, J. R. Campbell, and B. P.Gaber. 1995. Expression of the Neurospora crassa metallothionein gene in Escherichia coli and its effect on heavy-metal uptake. Appl. Microbiol. Biotechnol. 43:1112–1117.
Bae, W., W. Chen, A. Mulchandani, and R. Mehra. 2000. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins.Biotechnol. Bioeng. 70:518–523.
Chen, S., and D. B. Wilson. 1997. Construction and characterization ofEscherichia coli genetically engineered for bioremediation of Hg2-contaminatedenvironments. Appl. Environ. Microbiol. 63:2442–2445.
Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and theirpotential for Hg2 bioremediation. Biodegradation 8:97–103.
Ryan, J. P., M. C. Duncan, V. A. Bankaitis, and P. J. Bassford, Jr. 1986.Intragenic reversion mutations that improve export of maltose-binding protein in Escherichia coli malE signal sequence mutants. J. Biol. Chem. 261:3389–3395.
Kumamoto, C. A., and P. M. Gannon. 1988. Effects of Escherichia coli secBmutations on pre-maltose binding protein conformation and export kinetics.J. Biol. Chem. 263:11554–11558.
Liu, G., T. B. Topping, and L. L. Randall. 1989. Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein. Proc. Natl. Acad. Sci. USA. 86:9213–9217.
Clark F. Schierle, Mehmet Berkmen, Damon Huber, Carol Kumamoto, Dana Boyd, and Jon Beckwith. The DsbA Signal Sequence Directs Efficient, Cotranslational Export of Passenger Proteins to the Escherichia coli Periplasm via the Signal Recognition Particle Pathway
Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.
Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.