Difference between revisions of "Part:BBa K1130001"
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| + | ==Costa Rica 2022 Team Contribution== | ||
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| + | ===Usage and Biology=== | ||
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| + | 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). | ||
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| + | 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). | ||
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| + | ===References=== | ||
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| + | 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 | ||
Revision as of 08:13, 4 October 2022
Constitutive promoter 2 L.plantarum
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]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Costa Rica 2022 Team Contribution
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).
References
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