Difference between revisions of "Part:BBa K3924035"

 
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<partinfo>BBa_K3924035 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3924035 SequenceAndFeatures</partinfo>
  
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==Profile==
 +
Name: BSH1<br/>
 +
Base Pairs: 993bp<br/>
 +
Origin: Lactobacillus salivarius, synthetic<br/>
 +
Properties: A bile salt hydrolase catalyzing the hydrolysis of primary bile acids
 +
==Usage and Biology==
 +
BSH1 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, BSH1 has been successfully expressed in L.lactis and was shown to function well.<sup>[7]</sup>Based on these, we plan to express BSH1 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>
 +
==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/>
 +
In addition, to facilitate the verification of protein expression, a 6xHIS-tag was added to the N-terminal of BSH1 gene.
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[[Image: T--Tsinghua--BSH1_plasmid.png|center|600px|thumb|'''Figure 1: The design of BSH1''']]
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==Functional Verification==
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===Validation based on protein expression level===
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After sequencing of synthetic plasmid (by company), We transformed the plasmid into <i>E.coli</i> chemocompetent cells (Stellar, Takara) to amplify it. Then we transferred the plasmid to <i>Lactococcus lactis</i>. After overnight shaking culture, cells were harvested and lysed for westen blot. The result is shown below:
 +
[[Image: T--Tsinghua--BSH1_WesternBlot.png|center|600px|thumb|'''Figure 2: The Western Blot Result''']]
 +
The right lane, which was from the cell extract of <i>L.lactis</i> with pMG36e-BSH-1N, shows two bands at ~35kb and ~20kb that are not present in <i>L.lactis</i> without plasmid (left lane). We speculated that the 35kb band, in line with the molecular weight of BSH1, represents correctly expressed BSH1; the smaller 20kb band might be a truncated expression product, visible because it possesses 6xHis-tag at the N-terminal. However, due to the time limit, we did not further verify these speculations, and such results are admittedly not very convincing.
 +
 +
==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/>
 +
[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.<br/>
 +
[3] Begley, M., Hill, C., & Gahan, C. G. (2006). Bile salt hydrolase activity in probiotics. Applied and environmental microbiology, 72(3), 1729-1738.<br/>
 +
[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.<br/>
 +
[5] Nguyen, A., & Bouscarel, B. (2008). Bile acids and signal transduction: role in glucose homeostasis. Cellular signalling, 20(12), 2180-2197.<br/>
 +
[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.<br/>
 +
[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.<br/>
 +
[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.<br/>
  
 
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Latest revision as of 15:24, 21 October 2021


BSH1

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: BSH1
Base Pairs: 993bp
Origin: Lactobacillus salivarius, synthetic
Properties: A bile salt hydrolase catalyzing the hydrolysis of primary bile acids

Usage and Biology

BSH1 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, BSH1 has been successfully expressed in L.lactis and was shown to function well.[7]Based on these, we plan to express BSH1 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 BSH1 gene.

Figure 1: The design of BSH1

Functional Verification

Validation based on protein expression level

After sequencing of synthetic plasmid (by company), We transformed the plasmid into E.coli chemocompetent cells (Stellar, Takara) to amplify it. Then we transferred the plasmid to Lactococcus lactis. After overnight shaking culture, cells were harvested and lysed for westen blot. The result is shown below:

Figure 2: The Western Blot Result

The right lane, which was from the cell extract of L.lactis with pMG36e-BSH-1N, shows two bands at ~35kb and ~20kb that are not present in L.lactis without plasmid (left lane). We speculated that the 35kb band, in line with the molecular weight of BSH1, represents correctly expressed BSH1; the smaller 20kb band might be a truncated expression product, visible because it possesses 6xHis-tag at the N-terminal. However, due to the time limit, we did not further verify these speculations, and such results are admittedly not very convincing.

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.