Difference between revisions of "Part:BBa K2314831"

Revision as of 16:20, 1 November 2017

Pmini is a very short constitutive promoter in yeast, which is only 116bp, but with a good strength.

"Most of native yeast promoters can stretch hundreds of base pairs. Specifically, a single-gene circuit carrying a 1.5kb gene requires an additional 1kb of regulatory DNA (between the promoter and terminator) for appropriate expression, thus increasing the DNA cargo load by over 60%. However, this problem hasn’t been focused widely. With the development of synthetic biology, creating short but strong promoter is more important. Pmini is a short but strong constitutive promoter in yeast. It consists three main elements. Core element determines the shortest length required for transcription and serves as a platform for hybrid promoter technology. This core element scaffold was built on distinct, essential sequences for promoter function—a TATA box with consensus sequence of TATAWAWR24 followed by a transcription start site (TSS) with consensus sequence of A(Arich)5NYAWNN(Arich)6. UAS element contains transcription factor-binding sites (TFBS) and can aid in RNAP stabilization to enhanced transcription rates. The AT-rich spacer containing 30 nucleotides has a better performance.[1] We used Pmini promoter with synthetic terminator Tmini(BBa_K2314608) to construct a minimal genetic regulatory element “MINI-GRE“ combination. We also selected a commonly used promoter CYC1 and terminator CYC1, and contrusted four circuits to measure the performance of MINI-GRE combination. The fluorescent protein yECitrine (BBa_K2314024)was selected as the reporter protein. More details can be viewed in our project page.

[1]Redden H,Alper HS,The development and characterization of synthetic minimal yeast promoters[J],Nature Communication,2015,6 : 7810 "

In short，Pmini is a very short constitutive promoter in yeast, which is only 116bp in length, but with strong expression.

the structure of pmini:

the four circuits used in our project:

For convenience, we named the“CYC1p-yECitrine-CYC1t-mStrawberry-CYC1t”as“CC”,“CYC1p-yECitrine-MINIt-mStrawberry-CYC1t”as“CM”,“MINIp-yECitrine-CYC1t-mStrawberry-CYC1t”as“MC”, and“MINIp-yECitrine-MINIt-mStrawberry-CYC1t” as “MM”,hereafter.

The expression level of MINI-GRE:

The RT-PCR result of mini system:

Here, the transcript level of mStrawberry is very low, which means the transcription read through of MINI terminator can be overlooked. You can learn more from our project.

Sequence and Features