Difference between revisions of "20.109(S09):Prepare expression system (Day4)"
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==Introduction== | ==Introduction== | ||
− | Now that we have prepared DNA encoding your mutant inverse pericams, we would like to actually produce the physical proteins. Last time you were here, you transformed competent bacteria (called XL1-Blue) with mutagenized DNA prepared from a template plasmid. Successfully transformed bacteria grew into colonies on amipicillin-containing plates, and yesterday your oh-so devoted teaching staff picked two colonies per mutant to grow in liquid culture. The XL1-Blue cell line, although it now carries the inverse pericam DNA, cannot produce the inverse pericam protein. | + | Now that we have prepared DNA encoding your mutant inverse pericams, we would like to actually produce the physical proteins. Last time you were here, you transformed competent bacteria (called XL1-Blue) with mutagenized DNA prepared from a template plasmid. Successfully transformed bacteria grew into colonies on amipicillin-containing plates, and yesterday your oh-so devoted teaching staff picked two colonies per mutant to grow in liquid culture. The XL1-Blue cell line, although it now carries the inverse pericam DNA, cannot produce the inverse pericam protein. Today you will extract DNA from the XL1-Blue cells, prepare it for analysis, and transform your IPC mutant plasmids into a new bacterial system that ''can'' produce the protein directly. |
[[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 |