Part:BBa_K1941000:Experience
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Applications of BBa_K1941000
Repression of TEF1 promoter in yeasts
A) Repression design of Tef1 promoter. The hairpins PP7 or 2PP7 recruit the RNA binding domain PCP fused with the transcriptional repressor protein Mxi1. Then, dCas9 carries the repressor complex to the promoter Tef1, guided by the recognition sequence of the scRNA. B) Relative fluorescence histogram. GFP levels were measured using FACS after overnight cells growth. The data were normalized to report to the basal level: cells that have been integrated with a single plasmid carrying GFP under Tef1 promoter. A repression of 70 % GFP expression was noticeable with both constructs. C) Flow cytometry data (LSRII Snoopy) to quantify the level of GFP expression under the control of Tef1 promoter. In light blue, a population of cells that express dCas9, scTef1_PP7 and PCP-Mxi1. In grey, a population that express basal level of GFP under the control of Tef1 promoter.
Repression of CYC1 promoter in yeasts
B) A bar graph shows fluorescence intensity of a strain transformed with CYC1-GFP, scCYC1_PP7, the repressor fused protein PCP-Mxi1 and GAL1-dCas9. GAL1 promoter is usually used as the regulatory element to drive GAL-inducible genes. Therefore, when all components are present in cells but galactose is absent, dCas9 is not expressed On the other hand, when galactose is present GAL1 is activated and dCas9 is expressed. Cells were grown overnight in 2% glucose (blue) or galactose (red) selective media and GFP levels were measured using FACS. The data are displayed as mean ± standard deviation for 3 independent experiments. A 78% repression of GFP expression was noticeable under galactose induction. C) Flow cytometry data (LSRII Snoopy) quantify the level of GFP expression under the control of CYC1 promoter. Cells in bright blue were overnight cultured in a 2% galactose selective medium while dark blue in 2% glucose selective medium. As expected, cells grown in galactose clearly show a decreased level of GFP expression.
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