Part:BBa_K3612012
Pseudoalteromonas inducible promoter (pSHAB)
pSHAB is an inducible promoter from Pseudoalteromonas genus, which will be used for the AFPs expression in Pseudoalteromonas nigrifaciens.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1757
Illegal AgeI site found at 2129 - 1000COMPATIBLE WITH RFC[1000]
Usage
In recent years, the marine bacteria of the genus Pseudoalteromonas has been proposed as a non-conventional expression organism for low temperatures, due to its optimal growth in cold conditions and its ability to express “easy proteins” (1). With this purpose in mind, designed expression systems have successfully produced recombinant proteins in bacteria from this genus (2-4). Consequently, our team chose elements from these expression systems previously reported, like the pSHAB promoter, in order to control the expression of our protein type of interest, AFP, in Pseudoalteromonas nigrifaciens. This way, our team will use pSHAB along with its RBS (BBa_K3612030) and the reporter GFP (BBa_K3612010) to characterize the promoter, and with that, choose the most fit promoter for expression of the AFPs in Pseudoalteromonas nigrifaciens.
Biology
pSHAB is an inducible promoter coming from bacteria of the Pseudoalteromonas genus. Transcription with this promoter is low but in the presence of L-malate it has been shown to increase transcription of recombinant ꞵ-galactosidase to up to 13 times (5). L-malate induction occurs via a two-component regulatory sensor system. In this promoter there are two coding sequences: a putative C4-dicarboxylates sensor kinase (PSHAb0361 gene/MalS protein) and a putative C4 response regulator (PSHAb0362 gene/MalR protein) (5). L-malate directly promotes transcription of MalS; then, MalS acts as a phospho-donor for MalR, increasing the amount of the active form (6). Finally, the active form of MalR binds to the upstream region of the protein of interest and changes the transcription starting site to a more active one, highly increasing the expression (6).
References
(1) Sannino F, Giuliani M, Salvatore U, Apuzzo GA, de Pascale D, Fani R, et al. A novel synthetic medium and expression system for subzero growth and recombinant protein production in Pseudoalteromonas haloplanktis TAC125. Appl Microbiol Biotechnol. 2017 Jan 27;101(2):725–34.
(2) Parrilli E, Tutino ML. Psychrophiles: From Biodiversity to Biotechnology: Second Edition. Psychrophiles From Biodivers to Biotechnol Second Ed. 2017;1–685.
(3) Papa R, Rippa V, Sannia G, Marino G, Duilio A. An effective cold inducible expression system developed in Pseudoalteromonas haloplanktis TAC125. J Biotechnol. 2007 Jan 1;127(2):199–210.
(4) Yu ZC, Tang BL, Zhao DL, Pang X, Qin QL, Zhou BC, et al. Development of a cold-adapted Pseudoalteromonas expression system for the Pseudoalteromonas proteins intractable for the Escherichia coli system. PLoS One. 2015;10(9):1–14.
(5) Papa, R., Glagla, S., Danchin, A. et al. Proteomic identification of a two-component regulatory system in Pseudoalteromonas haloplanktis TAC125. Extremophiles. 2006; 10,483–491.
(6) Papa, R., Rippa, V., & Duilio, A. Identification of the transcription factor responsible for l-malate-dependent regulation in the marine Antarctic bacteriumPseudoalteromonas haloplanktis TAC125. FEMS Microbiology Letters. 2009;295(2), 177–186
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