Difference between revisions of "Part:BBa K2314831"

Latest revision as of 19:22, 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 TATAWAWR followed by a transcription start site (TSS) with consensus sequence of A(A_rich)₅NYAWNN(A_rich)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 MINI promoter：

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:

Abs600 of strains with different promoter-terminator pairs.png

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

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]

@@ Line 3: / Line 3: @@
 "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]
+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 TATAWAWR followed by a transcription start site (TSS) with consensus sequence of  A(A<sub>rich</sub>)<sub>5</sub>NYAWNN(A<sub>rich</sub>)<sub>6</sub>. 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.
@@ Line 15: / Line 15: @@
-the structure of pmini:
+The structure of MINI promoter：
 https://static.igem.org/mediawiki/parts/1/18/T--OUC-China--MINIP.jpg
-the four circuits used in our project:
+The four circuits used in our project:
-[[Image:T--OUC-China--MINI-GRE_CC_PLASMID.png|200px|thumb|center|UTRrpsT enhances protein expression by structure at the 5’UTR.]]
+[[Image:T--OUC-China--MINI-GRE_CC_PLASMID.png|800px|thumb|center]]
+[[Image:T--OUC-China--MINI-GRE_CM_PLASMID.png|800px|thumb|center]]
+[[Image:T--OUC-China--MINI-GRE_MC_PLASMID.png|800px|thumb|center]]
+[[Image:T--OUC-China--MINI-GRE_MM_PLASMID.png|800px|thumb|center]]
-https://static.igem.org/mediawiki/parts/d/d6/T--OUC-China--MINI-GRE_CC_PLASMID.png
-https://static.igem.org/mediawiki/parts/c/ca/T--OUC-China--MINI-GRE_CM_PLASMID.png
+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.
-https://static.igem.org/mediawiki/parts/b/bc/T--OUC-China--MINI-GRE_MC_PLASMID.png
-https://static.igem.org/mediawiki/parts/1/10/T--OUC-China--MINI-GRE_MM_PLASMID.png
+The expression level of MINI-GRE:
+[[Image:Abs600_of_strains_with_different_promoter-terminator_pairs.png|800px|thumb|center]]
-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 RT-PCR result of mini system:
+[[Image:T--OUC-China--MINI-GRE_qPCR_.png|800px|thumb|center]]
-The expression level 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.
-https://static.igem.org/mediawiki/parts/f/f4/Abs600_of_strains_with_different_promoter-terminator_pairs.png
-The RT-PCR result of mini system:
-https://static.igem.org/mediawiki/parts/9/97/T--OUC-China--MINI-GRE_qPCR_.png
-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.