Part:BBa_K4788005

IsmA-BSH gene

Production of bile salt lyase while converting cholesterol into 4-cholesten-3-one

Usage and Biology

It is a part composed of the IsmA gene and the BSH gene, which can directly degrade cholesterol. The BSH gene can indirectly regulate cholesterol by producing bile salt lyase. Their combination can have a corresponding effect on cholesterol regulation.

Characterization

In order to characterize the corresponding components, we need to develop different characterization schemes for different genes. For the BSH gene, we used MRS bile salt qualitative plate inoculation experiments to characterize it, and for the IsmA gene, we used the OPA method to detect cholesterol residue after cholesterol culture medium cultivation to characterize it. Meanwhile, as short chain fatty acids are also important cholesterol regulatory substances, we can also construct the IsmA-BSH-BCoAT triplet gene to achieve more complex cholesterol regulation. For more information on triplets, please refer to BBa_K4788007. In the characterization of the triad, as shown in Figure 1, we can see that the IsmA gene in the I-BS gene has been successfully characterized.

Figure1: a) Cholesterol residue in the culture medium after cultivation of engineering bacteria on basic cholesterol medium b) Percentage of cholesterol degradation in the culture medium after cultivation of engineering bacteria on basic cholesterol medium

As for the BSH gene characterization of the dimer (IsmA-BSH),the BSH characterization of the dimer in the experimental images of the above part is not good, but in another set of experiments we can see it more clearly in Figure 2.

Figure2: Binary BSH qualitative plate

Reference

Jia B, Zou Y, Han X, Bae JW, Jeon CO. Gut microbiome-mediated mechanisms for reducing cholesterol levels: implications for ameliorating cardiovascular disease. Trends Microbiol. 2023 Jan;31(1):76-91. doi: 10.1016/j.tim.2022.08.003. Epub 2022 Aug 22. PMID: 36008191. Kenny DJ, Plichta DR, Shungin D, Koppel N, Hall AB, Fu B, Vasan RS, Shaw SY, Vlamakis H, Balskus EP, Xavier RJ. Cholesterol Metabolism by Uncultured Human Gut Bacteria Influences Host Cholesterol Level. Cell Host Microbe. 2020 Aug 12;28(2):245-257.e6. doi: 10.1016/j.chom.2020.05.013. Epub 2020 Jun 15. PMID: 32544460; PMCID: PMC7435688.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1772
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 58
  
• 1000
  COMPATIBLE WITH RFC[1000]