Difference between revisions of "Part:BBa K3924036"
Yiyuan-h19 (Talk | contribs) |
(→Design and Construction) |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 14: | Line 14: | ||
Name: BSH2<br/> | Name: BSH2<br/> | ||
Base Pairs: 996bp<br/> | Base Pairs: 996bp<br/> | ||
| − | Origin: Lactobacillus salivarius, synthetic<br/> | + | Origin: <i>Lactobacillus salivarius</i>, synthetic<br/> |
Properties: A bile salt hydrolase catalyzing the hydrolysis of primary bile acids | Properties: A bile salt hydrolase catalyzing the hydrolysis of primary bile acids | ||
| + | |||
==Usage and Biology== | ==Usage and Biology== | ||
| − | BSH2 is a bile salt hydrolase from Lactobacillus salivarius. It catalyzes deconjugation of glyco-conjugated and tauro-conjugated bile acids through the hydrolysis of the amide bond and the release of free bile acids (e.g. cholic acid, deoxycholic acid or chenodeoxycholic acid) and amino acids (glycine or taurine)<sup>[1]</sup>. Deconjugation is a rate-limiting reaction in the metabolism of bile acids in the small intestine<sup>[2][3]</sup>. Therefore, BSH participates in a range of metabolic processes in mammalians including the regulation of dietary lipid absorption, cholesterol metabolism, energy and inflammation homeostasis.<sup>[4][5][6]</sup>According to existing literature, BSH2 has been successfully expressed in L.lactis and was shown to function well.<sup>[7]</sup>Based on these, we plans to express BSH2 in the well-characterized probiotic strain Lactococcus lactis to hydrolyse primary bile acids in the intestine, promoting the production of the more benefitial secondary bile acids. <sup>[8]</sup> | + | BSH2 is a bile salt hydrolase from <i>Lactobacillus salivarius</i>. It catalyzes deconjugation of glyco-conjugated and tauro-conjugated bile acids through the hydrolysis of the amide bond and the release of free bile acids (e.g. cholic acid, deoxycholic acid or chenodeoxycholic acid) and amino acids (glycine or taurine)<sup>[1]</sup>. Deconjugation is a rate-limiting reaction in the metabolism of bile acids in the small intestine<sup>[2][3]</sup>. Therefore, BSH participates in a range of metabolic processes in mammalians including the regulation of dietary lipid absorption, cholesterol metabolism, energy and inflammation homeostasis.<sup>[4][5][6]</sup>According to existing literature, BSH2 has been successfully expressed in <i>L.lactis</i> and was shown to function well.<sup>[7]</sup>Based on these, we plans to express BSH2 in the well-characterized probiotic strain <i>Lactococcus lactis</i> to hydrolyse primary bile acids in the intestine, promoting the production of the more benefitial secondary bile acids. <sup>[8]</sup> |
| + | |||
==Design and Construction== | ==Design and Construction== | ||
| − | As our engineered probiotics are to be used in IBD treatment as prescribed drugs instead of dietary supplements, we deem it unnecessary to incorporate conditional promoters into our design. We therefore use the constitutive expression plasmid pMG36e as the backbone. Also, being a transfer plasmid, pMG36e can replicate in both E.coli and L.lactis, which saved us a lot of trouble in transformation.<br/> | + | As our engineered probiotics are to be used in IBD treatment as prescribed drugs instead of dietary supplements, we deem it unnecessary to incorporate conditional promoters into our design. We therefore use the constitutive expression plasmid pMG36e as the backbone. Also, being a transfer plasmid, pMG36e can replicate in both <i>E.coli</i> and <i>L.lactis</i>, which saved us a lot of trouble in transformation.<br/> |
In addition, to facilitate the verification of protein expression, a 6xHIS-tag was added to the N-terminal of BSH2 gene. | In addition, to facilitate the verification of protein expression, a 6xHIS-tag was added to the N-terminal of BSH2 gene. | ||
[[Image: T--Tsinghua--BSH2_plasmid.png|center|600px|thumb|'''Figure 1: The design of BSH2''']] | [[Image: T--Tsinghua--BSH2_plasmid.png|center|600px|thumb|'''Figure 1: The design of BSH2''']] | ||
| + | |||
==Reference== | ==Reference== | ||
[1] Grill, J., Schneider, F., Crociani, J., & Ballongue, J. (1995). Purification and characterization of conjugated bile salt hydrolase from Bifidobacterium longum BB536. Applied and environmental microbiology, 61(7), 2577-2582.<br/> | [1] Grill, J., Schneider, F., Crociani, J., & Ballongue, J. (1995). Purification and characterization of conjugated bile salt hydrolase from Bifidobacterium longum BB536. Applied and environmental microbiology, 61(7), 2577-2582.<br/> | ||
Latest revision as of 15:21, 21 October 2021
BSH2
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 316
Illegal EcoRI site found at 538 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 316
Illegal EcoRI site found at 538 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 316
Illegal EcoRI site found at 538 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 316
Illegal EcoRI site found at 538 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 316
Illegal EcoRI site found at 538 - 1000COMPATIBLE WITH RFC[1000]
Profile
Name: BSH2
Base Pairs: 996bp
Origin: Lactobacillus salivarius, synthetic
Properties: A bile salt hydrolase catalyzing the hydrolysis of primary bile acids
Usage and Biology
BSH2 is a bile salt hydrolase from Lactobacillus salivarius. It catalyzes deconjugation of glyco-conjugated and tauro-conjugated bile acids through the hydrolysis of the amide bond and the release of free bile acids (e.g. cholic acid, deoxycholic acid or chenodeoxycholic acid) and amino acids (glycine or taurine)[1]. Deconjugation is a rate-limiting reaction in the metabolism of bile acids in the small intestine[2][3]. Therefore, BSH participates in a range of metabolic processes in mammalians including the regulation of dietary lipid absorption, cholesterol metabolism, energy and inflammation homeostasis.[4][5][6]According to existing literature, BSH2 has been successfully expressed in L.lactis and was shown to function well.[7]Based on these, we plans to express BSH2 in the well-characterized probiotic strain Lactococcus lactis to hydrolyse primary bile acids in the intestine, promoting the production of the more benefitial secondary bile acids. [8]
Design and Construction
As our engineered probiotics are to be used in IBD treatment as prescribed drugs instead of dietary supplements, we deem it unnecessary to incorporate conditional promoters into our design. We therefore use the constitutive expression plasmid pMG36e as the backbone. Also, being a transfer plasmid, pMG36e can replicate in both E.coli and L.lactis, which saved us a lot of trouble in transformation.
In addition, to facilitate the verification of protein expression, a 6xHIS-tag was added to the N-terminal of BSH2 gene.
Reference
[1] Grill, J., Schneider, F., Crociani, J., & Ballongue, J. (1995). Purification and characterization of conjugated bile salt hydrolase from Bifidobacterium longum BB536. Applied and environmental microbiology, 61(7), 2577-2582.
[2] Geng, W., & Lin, J. (2016). Bacterial bile salt hydrolase: an intestinal microbiome target for enhanced animal health. Animal health research reviews, 17(2), 148-158.
[3] Begley, M., Hill, C., & Gahan, C. G. (2006). Bile salt hydrolase activity in probiotics. Applied and environmental microbiology, 72(3), 1729-1738.
[4] Joyce, S. A., Shanahan, F., Hill, C., & Gahan, C. G. (2014). Bacterial bile salt hydrolase in host metabolism: potential for influencing gastrointestinal microbe-host crosstalk. Gut microbes, 5(5), 669-674.
[5] Nguyen, A., & Bouscarel, B. (2008). Bile acids and signal transduction: role in glucose homeostasis. Cellular signalling, 20(12), 2180-2197.
[6] Yadav, R., & Shukla, P. (2017). An overview of advanced technologies for selection of probiotics and their expediency: a review. Critical reviews in food science and nutrition, 57(15), 3233-3242.
[7] Lavelle, A., & Sokol, H. (2020). Gut microbiota-derived metabolites as key actors in inflammatory bowel disease. Nature reviews Gastroenterology & hepatology, 17(4), 223-237.
[8] Bi, J., Liu, S., Du, G., & Chen, J. (2016). Bile salt tolerance of Lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells. Biotechnology letters, 38(4), 659-665.