Part:BBa_K2559003
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.
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!
GZHS-United
We linked the gene of ATAPX1 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.
< img src="https://static.igem.org/mediawiki/parts/7/7a/HS_60_biobricks.png" width="550" height="300" BORDER=0
To test the heat-sensitive construction, an overnight culture of our E.coli strain carrying the AsRed2 gene under the control of groE promoter was set up at 25 ºC. After that, OD was adjusted to 0.15 and several aliquots were transferred to fluorimetry cuvettes. Heat shock was carried out at different temperatures in a water bath. Then, the cuvettes were maintained at room temperature and OD and fluorescence intensity were measured at different time points.
As shown in the graphs below, we found a correlation between the temperature at which heat shock was carried out and the level of expression of the fluorescent prote
You can see more information in our model page! ( http://2019.igem.org/Team:GZHS-United/Model)
Sequence and Features
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