Difference between revisions of "20.109(S09):Prepare expression system (Day4)"
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[[Image:20.109_pREST.png|thumb|right|From Invitrogen manual]] | [[Image:20.109_pREST.png|thumb|right|From Invitrogen manual]] | ||
− | The bacterial expression vector we are using [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=615 (pRSET)] contains the bacteriophage T7 promoter. This promoter is active only in the presence of T7 RNA polymerase (T7RNAP), an enzyme that therefore must be expressed by the bacterial strain used to make the protein of interest. We will use the BL21(DE3)pLysS strain, which has the following genotype: F<sup>-</sup>, ''omp''T ''hsd''SB (r<sub>B</sub><sup>-</sup> m<sub>B</sub><sup>-</sup>) ''gal dcm'' (DE3) pLysS (Cam<sup>R</sup>). In BL21(DE3), T7RNAP is associated with a ''lac'' construct, and its expression is under the control of the ''lac''UV5 promoter. Due to the action of the ''lac'' repressor (''lac''I gene), the polymerase will not be produced except in the presence of lactose or a small-molecule lactose analogue such as IPTG (isopropyl β-D-thiogalactoside). To further reduce | + | The bacterial expression vector we are using [https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=615 (pRSET)] contains the bacteriophage T7 promoter. This promoter is active only in the presence of T7 RNA polymerase (T7RNAP), an enzyme that therefore must be expressed by the bacterial strain used to make the protein of interest. We will use the BL21(DE3)pLysS strain, which has the following genotype: F<sup>-</sup>, ''omp''T ''hsd''SB (r<sub>B</sub><sup>-</sup> m<sub>B</sub><sup>-</sup>) ''gal dcm'' (DE3) pLysS (Cam<sup>R</sup>). In BL21(DE3), T7RNAP is associated with a ''lac'' construct, and its expression is under the control of the ''lac''UV5 promoter. Due to the action of the ''lac'' repressor (''lac''I gene), the polymerase will not be produced except in the presence of lactose or a small-molecule lactose analogue such as IPTG (isopropyl β-D-thiogalactoside). To further reduce |