Part:BBa_K4133021
P48 + RBS_tuf33 + GFP + Double Terminator
This part was created by combining the following parts: A P48 constitutive promoter (BBa_K1130001) a a L. plantarum RBS (BBa_K4133004), a GFP codon optimized for L. casei (BBa_K4133005) and the double terminator T7 and T1rrb (BBa_K3183021). The combination of these parts was performed with the aim to characterize the influence of different regulatory sequences and their combination on recombinant gene expression in Lactobacillus casei ATCC 393.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 709
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 79
Usage and Biology
The p48 synthetic promoter sequence was derived from consensus sequences obtained from the 16S rRNA promoter alignment, using the L. plantarum WCFS1 genome, as part of a constitutive promoter library for L. plantarum developed by Rud and collaborators (2006). This sequence was obtained following the methodology of Jensen & Hammer (1998), where the spacer sequence separating the promoter consensus sequences is randomized. It should be noted that spacer sequences can affect the three-dimensional structure of the promoter, thus affecting the interaction between the RNA polymerase and the promoter. In this way, to obtain the P48 promoter, a PCR was performed with the pSIP vector and an oligonucleotide containing the consensus sequences and randomized spacer sequences as a forward primer to amplify the gusA reporter protein from the pSIP409 plasmid. The resulting PCR amplicon, containing the P48 synthetic promoter followed by the gusA reporter gene, was inserted and characterized in the pSIP409 vector (Rud et al., 2006).
Among the advantages of using a constitutive promoter such as p48 is their potential to obtain a stable yield easily and to perform metabolic control analysis. Also, because this promoter is reported to be strong, it can be used for high-level protein production. By using constitutive promoters, there is also the advantage of not depending on the addition of an inducer to the medium for activation, which can generate high costs in large-scale fermentations. Finally, the use of constitutive promoters can be considered for the production of antigens for oral vaccination purposes, taking advantage of the probiotic potential of Lactobacillus species (Rud et al., 2006).
Characterization
In order to characterize the function of this promoter, it was combined with the RBSs BBa_K4133004 and BBa_K2760006, giving rise to the composite parts BBa_K4133021 and BBa_K4133022 respectively, which uses a Green Fluorescent Protein as reporter gene BBa_K4133005. Thus, both BBa_K4133021 and BBa_K4133022 were inserted into our pLBov plasmid (BBa_K4133000), for subsequent transformation into E. coli DH5ⲁ and L. casei ATCC 393. But once the transformed E. coli colonies were grown, we observed that some transformed with BBa_K4133021 had a different coloration from the rest, so we proceeded to observe this plate under UV light. Thus, when we exposed the plate to UV light, we observed that there were indeed E. coli colonies transformed with pLBov + BBa_K4133021 that were able to emit fluorescence (Figure 1), i.e., both the p48 promoter and our RBS_pTuf33 allowed expression of a GFP, which in turn was originally codon-optimized for L. casei.
Figure 1. E. coli DH5ⲁ transformed with pLBov + BBa_K4133021 emitting fluorescence under UV light.
References
Jensen, P. R., & Hammer, K. (1998). The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Applied and environmental microbiology, 64(1), 82-87.
Rud, I., Jensen, P. R., Naterstad, K., & Axelsson, L. (2006). A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiology, 152(4), 1011–1019. https://doi.org/10.1099/mic.0.28599-0
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