Part:BBa_K4988003
PfadBA mutant pAR+IsmA
Considering the fact that after the stimulation of oleic acid, oleic acid promoter can activate the expression of the downstream gene which ensure the stability and efficiency of the expression we decided to use oleic acid promoter as our promoter. Additionally, IsmA gene are effective in transforming and degrading cholesterol, we decided to use oleic acid to activate its function. Oleic acid promoter is derived from Escherichia coli, and we use its high efficiency mutation pAR derived from pfadBA. iSMA, derived from human gut microbes, mediates the degradation of cholesterol to coprosterol.
Usage and Biology
We use oleic acid promoter to express IsmA gene and transform the plasmids into E.coli Rosetta.
Figure 1 Design of oleic acid promoter and IsmA.
Characterization
The LB medium was added with different concentrations of oleic acid and inoculated with E.coli Rosetta. The OD600 value of E.coli Rosetta was measured by spectrophotometer. The results showed that 5 mM oleic acid had little effect on bacterial growth, while 10 mM oleic acid inhibited bacterial growth.
Figure 2 Gel electrophoresis of ismA and map of recombinant plasmid pSB1A3-pAR-ismA.
Figure3 Effect of oleic acid on bacterial growth.
The LB medium was added with different concentrations of oleic acid and inoculated with E.coli Rosetta/pOleic ACID-MRFP. Adjust the initial OD600 to 0.1. After 12 hours, the OD600 value and mRFP fluorescence intensity were measured using an microplate reader. The results showed that under the induction of oleic acid at 0mM and 5 mM, oleic acid promoter mediated the engineering bacteria to produce about 4.5 Fold change under the induction of 5mM oleic acid comparing to the result produced by omM oleic acid.
Figure4 Response of oleic acid promoter.
The ismA gene was cloned into the pET28a plasmid and validated before being transformed into Escherichia coli Rosetta. The recombinant Rosetta bacteria were then cultured in LB medium containing 0.5 mM IPTG and incubated at 16°C for 12 hours. A 10 mL bacterial culture was taken, centrifuged to remove the supernatant, and the cell pellet was resuspended in 20 mM Tris-HCl (pH 7.0). The cells were then subjected to ice-cold sonication to collect the crude enzyme solution, and the protein concentration was determined using the Bradford assay. The crude enzyme solution was mixed with 5 mg/mL cholesterol and incubated for 12 hours. Subsequently, the cholesterol concentration was determined by measuring the fluorescence emission using the Amplex™Red Cholesterol Assay Kit. The results, as shown in the figure, indicate that the engineered strain significantly degraded cholesterol compared to the wild-type.
Figure 5 The expression of ismA.
The recombinant strain Rosetta was then placed in LB medium containing different concentrations of oleic acid. After 12 hours, the crude enzyme was extracted according to the above method. 5 mg/mL crude enzyme solution was incubated with 200 μM cholesterol for 12 h. The Amplex™Red cholesterol test kit (Sigma, A12216) was then used to determine cholesterol concentrations based on fluorescence emission. Under the induction of 5mM of oleic acid , the cholesterol concentration goes through a dramatic decrease due to the expression of IsmA gene, comparing to the concentration when omM oleic acid is present. The result shows oleic acid can stimulate the oleic acid promoter to express IsmA gene which can then lower cholesterol concentration.
Figure6 The expression of ismA is controlled by oleic acid promoter.
Potential application directions
Experiments shows that oleic acid can effectively activate the oleic acid promoter which then stimulate the IsmA gene and decrease the cholesterol concentration considerably. As a result, oleic acid promoter is going to be useful in producing low cholesterol foods and helps decrease the cholesterol intake of potential high cholesterol patients and helps decrease the risk of diseases such as cardiovascular disease caused by high cholesterol. Oleic acid promoter can be used as a clinical treatment for high cholesterol in the future and also provide new ideas and solutions.
References
1.Ma, Yueyuan & Zheng, Xiangrui & Lin, Yi-Na & Zhang, Lizhan & Yuan, Yiping & Wang, Huan & Winterburn, James & Wu, Fuqing & Wu, Qiong & Ye, Jian-Wen & Chen, Guo-Qiang. (2022). Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas. Metabolic Engineering. 72. 10.1016/j.ymben.2022.04.003. 2.Yueyuan Ma, Xiangrui Zheng, Yina Lin, Lizhan Zhang, Yiping Yuan, Huan Wang, James Winterburn, Fuqing Wu, Qiong Wu, Jian-Wen Ye, Guo-Qiang Chen,Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas,Metabolic Engineering,Volume 72,2022,Pages 325-336. 3.Zhang, Fuzhong, James M. Carothers, and Jay D. Keasling. "Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids." Nature biotechnology 30.4 (2012): 354-359. 4.Tok, Esra, and Belma Aslim. "Cholesterol removal by some lactic acid bacteria that can be used as probiotic." Microbiology and immunology 54.5 (2010): 257-264.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 140
- 1000COMPATIBLE WITH RFC[1000]
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