Difference between revisions of "Part:BBa K3171175"

Revision as of 16:28, 20 October 2019

pTet-mCherry optimized in E. coli

Enhanced pTET mCherry optimized for E. coli. Improvisation of the pTET promoter to enhance its strength in E. coli. Using a synthetic promoter library on pTet (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 improved pTET promoter can be found at, Ba_K3171173. 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K3171175 short</partinfo>
-Enhanced pTET mCherry for E. coli
+Enhanced pTET mCherry optimized for E. coli. Improvisation of the pTET promoter to enhance its strength in E. coli. Using a synthetic promoter library on pTet (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 improved pTET promoter can be found at, [https://parts.igem.org/Part:BBa_K3171173B Ba_K3171173].
+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.
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
-Improvisation of the pTET promoter to enhance its strength in E. coli. Using a synthetic promoter library on pTet (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. 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. This promoter is also used in the composite part with mCherry fusion protein.