Difference between revisions of "Part:BBa K3171173"
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| − | Improvisation of the pTET promoter to enhance its strength in E. coli. Using a synthetic promoter library on pTet | + | Improvisation of the pTET promoter to enhance its strength in E. coli. Using a synthetic promoter library on pTet [[Part:BBa_R0040]] in order to enhance function and inducibility. |
In order to enhance function and inducibility, we optimized tetracycline-inducibility in E. coli by creating randomizations in the sequence to obtain arbitrary promoters. In order to improve the pTET promoter, a construct of pTET-mCherry was designed using 3A assembly and cloned in E. coli. The cells were induced with 125, 250 and 500ng/mL anhydrous tetracycline. The expression of mCherry was determined with the help of the fluorescence level emitted by it. These fluorescence levels were measured at different time points of 0, 4, 8 and 12 hours after induction. The screening was done based on quantifications of the fluorescence intensity measured. The best version of the promoter was determined based on fold change in the fluorescence intensity and low basal level. | In order to enhance function and inducibility, we optimized tetracycline-inducibility in E. coli by creating randomizations in the sequence to obtain arbitrary promoters. In order to improve the pTET promoter, a construct of pTET-mCherry was designed using 3A assembly and cloned in E. coli. The cells were induced with 125, 250 and 500ng/mL anhydrous tetracycline. The expression of mCherry was determined with the help of the fluorescence level emitted by it. These fluorescence levels were measured at different time points of 0, 4, 8 and 12 hours after induction. The screening was done based on quantifications of the fluorescence intensity measured. The best version of the promoter was determined based on fold change in the fluorescence intensity and low basal level. | ||
Revision as of 18:20, 20 October 2019
Improved pTet promoter in E. coli
Improvisation of the pTET promoter to enhance its strength in E. coli. Using a synthetic promoter library on pTet Part:BBa_R0040 in order to enhance function and inducibility. In order to enhance function and inducibility, we optimized tetracycline-inducibility in E. coli by creating randomizations in the sequence to obtain arbitrary promoters. In order to improve the pTET promoter, a construct of pTET-mCherry was designed using 3A assembly and cloned in E. coli. The cells were induced with 125, 250 and 500ng/mL anhydrous tetracycline. The expression of mCherry was determined with the help of the fluorescence level emitted by it. These fluorescence levels were measured at different time points of 0, 4, 8 and 12 hours after induction. The screening was done based on quantifications of the fluorescence intensity measured. The best version of the promoter was determined based on fold change in the fluorescence intensity and low basal level.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]