Part:BBa_K4727007:Design
Pf3 Pseudomonas Phage Gene 3 expression cassette
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 657
Illegal BsaI site found at 981
Design Notes
A strong but inducible promoter was chosen; this allows the metabolic burden to be limited, if necessary, while, when fully induced, enabling high expression levels of the alternative gene so that it can be preferentially incorporated into the phage particles. The selected promoter was pL-lac0-1 (BBa_J428041) that combines the strength of the pL promoter, with the possibility to regulate it, derived from the LacO binding site. Indeed, if the cell expresses the LacI gene at high levels, it becomes feasible to induce the expression of the gene regulated by this promoter through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium. Moreover, by employing varying concentrations of the inducer, it is possible to finely adjust the expression level, thus creating a controllable system that is in line with the requirement of promoting cell vitality. Similarly, the choice of the ribosome binding site (RBS) fell on a strong element to ensure that, upon transcript production, it is efficiently translated by ribosomes. This decision aligns with the aim of favoring the production and incorporation of this gene product over the gene 3 of phage M13. Hence, the RBS chosen was BBa_B0034. Finally, the terminator was selected based on its strength and efficiency, with a preference for an intrinsic terminator, independent from the expression of cellular proteins. Therefore, the T1 terminator of E. coli was opted (part BBa_B0010).
Source
Pf3 bacteriophage deposited genome NCBI ID: NC_074763 Registty of Standard biological parts