Difference between revisions of "Part:BBa K2559003"

Revision as of 19:39, 18 October 2019

Ecoli promoter of sgRNA(PrplJ)

The BBa_K2559003 , PrplJ promoter is a strong endogenous promoter in Escherichia coli K 12. We used it to express single guide RNA in our CRISPR/cas9 system.

Usage and Biology

We obtained this promoter from a database named PromEC ( http://margalit.huji.ac.il/promec/index.html) .PromEC is an updated compilation of E. coli mRNA promoter sequences. It includes detailed information of genesites which have been experimentally identified mRNA transcriptional starting position in the E. coli chromosome, as well as the actual sequences of the promoters. We have built up a model to measure the expression intensity of these promoters. Eventually, we selected three promoters including: PrplJ, Pdapa and PcaiF based on the results of modeling PrplJ, Pdapa and PcaiF.We further made chimeric fluorescent fusion proteins between the promoter and GFP respectively and tested the fluorescent intensity driven by different promoters. The combination of PromEC and our modeling can be used as a efficient tool to figure out the information about interested promoters. The GFP expression in E.coli (DH5 α) and was driven by PrplJ, Pdapa and PcaiF promoter.

Figure1 The result of the fluorescent intensity measurement

Figure2 Fluorescent intensity of eGFP driven by by PrplJ, PdapA, PcaiF promoter.

Basing on the model predition and experiment testing results,we used PrplJ as the promoter of sgRNA

The part is facilitate the in-depth research for other teams!

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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 The part is facilitate the in-depth research for other teams!
-===GZHS-United===
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-We linked the gene of arabidopsis APX to the existing promoter PrplJ, measured its enzyme activity in different states (different temperature, different PH, different light), and obtained correlation results between different factors and expressed enzyme protein activity.
-Data which are obtained by our results, we verify of the type of the promoter that PrplJ stability and reusability, in the experimental projects require different promoter can play a good role, such as the need to build contains multiple promoter and the need to use Gisbon connection, in order to prevent the homologous recombination failure, must use a different promoter, PrplJ is a reliable choice.