Parts
Porine-pIV leaky-Marciano : BBa_K2093000
The BioBrick that we submitted encodes a mutated version of the pIV porin from the filamentous bacteriophage f1. Those secretins are quite interesting since their opening are controlled by phages (1). When expressed in E. coli, this porin is localized in the outer membrane. The mutation confers a "leaky" phenotype to the bacteria, i.e. periplasmic content is released in the culture supernatant (2). Spagnuolo and his team used a random mutation technique, followed by a positive selection to get those leaky mutants. Indeed mutants having trouble to stay entirely closed or opened (leaky), were obtained after mutations in the C-terminal secretin family domain; GATE 1 and GATE 2 and in the N-terminal (periplasmic portion) which could be involved in the controlled opening of the gate.
This property is potentially useful for secreting recombinant proteins or metabolites. When expressed in E. coli strains lacking the endogenous lamB outer membrane protein, the pIV allows the bacteria to grow on oligomaltosides (tetra-, penta-, hexa- and hepta-maltose) as carbon source.
The part is expected to work under different promoters. Furthermore this part contain a sequence which allows the protein to be inserted in the outer membrane of the bacteria.
To get more information about this pIV-leaky-Marciano part you can check the following articles.
Spagnuolo, J., Opalka, N., Wen, W. X., Gagic, D., Chabaud, E., Bellini, P., … Rakonjac, J. (2010). Identification of the gate regions in the primary structure of the secretin pIV. Molecular Microbiology, 76(1), 133–150. doi:10.1111/j.1365-2958.2010.07085.x
Marciano, Denise K., Marjorie Russel, et Sanford M. Simon. 1999. « An Aqueous Channel for Filamentous Phage Export ». Science 284 (5419): 1516‑19. doi:10.1126/science.284.5419.1516.
BioBrick construction
As required, we submit our PIV-leaky-Marciano cloned in pSB1C3 plasmid.
This high copy number plasmid backbone is carrying a chloramphenicol resistance gene and can easily be sequenced by the iGEM labs.
We received it in the iGEM kit, linearized. As we tried to amplify it in E. coli TOP 10 culture we had the pleasure to visualize the expression of the monomeric Red Fluorescence Protein 1 which is replaceable by our Part.
Once our part in inserted between the two restriction sites, colonies recover a “normal” colour.
The figure below represents both maps of the pSB1C3 plasmid without and with our Part, the pIV-leaky-Marciano:
Here is the simplified protocol that we used before sending our Part:
Amplifying pSB1C3
- Electroporation of pSB1C3 in E.coli TOP10
- Isolation of the pSB1C3 plasmid
- Restriction
- Electrophoresis gel
Cloning the PIV-Leaky-Marciano Part in pSB1C3
- Restriction of the pSB1C3 plasmid
- PCR of the PIV leaky sequence
- Digestion of the PCR product
- Purifying and restriction of the PCR product and the pSB1C3
- Insertion/Ligation
How is the part is expected to work ?
This BioBrick is expected to work under a variety of promoters such as pPro 30. This Part will provide you a way of secreting aspecific small peptides from the periplasm to the extracellular media.
This has been done before by UCL labs using cyclic peptides. To get more information about what you could do with this part, please check the following article.
Deschuyteneer, G., Garcia, S., Michiels, B., Baudoux, B., Degand, H., Morsomme, P., & Soumillion, P. (2010). Intein-mediated cyclization of randomized peptides in the periplasm of Escherichia coli and their extracellular secretion. ACS Chemical Biology, 5(7), 691–700. doi:10.1021/cb100072u
Bibliography
Deschuyteneer, G., Garcia, S., Michiels, B., Baudoux, B., Degand, H., Morsomme, P., & Soumillion, P. (2010). Intein-mediated cyclization of randomized peptides in the periplasm of Escherichia coli and their extracellular secretion. ACS Chemical Biology, 5(7), 691–700. doi:10.1021/cb100072u
Marciano, Denise K., Marjorie Russel, et Sanford M. Simon. 1999. « An Aqueous Channel for Filamentous Phage Export ». Science 284 (5419): 1516‑19. doi:10.1126/science.284.5419.1516.
Spagnuolo, J., Opalka, N., Wen, W. X., Gagic, D., Chabaud, E., Bellini, P., … Rakonjac, J. (2010). Identification of the gate regions in the primary structure of the secretin pIV. Molecular Microbiology, 76(1), 133–150. doi:10.1111/j.1365-2958.2010.07085.x
