Part:BBa_K1639009
LacI regulated DsRed with miR 26a and miR 375 binding sites
Usage and Biology
In our project Cancer module consist of three parts. First one produces tTA to activate expression of mLacI protein form pTRE. "m" in mLacI stands for mammalian, it's optimized version of bacterial LacI for expression in eucaryots.Produced mLacI would suppress CMV IE promoter through lac operators.(Figure 1)
This part composed of two lac operators which followed up with DsRed protein (red flourescent protein derived from Discosoma spp) and miRNA binding sites at 3'end. This miRNA binding sites are complementary to low-miRNA in gastric cancer cells. In normal cells the level of these miRNas are higher than in gastric cancerous cells they would suppress expression of DsRed protein.
Cloning and Expression
pTRE-gene ligation
Our plan was fistly to clone this part in gBlock form into pTRE vector. For this we used EcoR1 and BamH1 enzymes, after succesfull cloning colony PCR and cut-checks were performed for verification. (Figure 2)
CMV IE ligation with pTRE-gene
Once we successfully cloned pTRE- LacO2-DsRed-miR375-miR26a bs, we needed to change the minimal CMV promoter found in pTRE with the CMV IE promoter.To achieve this, we cut the CMV IE promoter from the pTET off plasmid using XhoI and EcoRI enzymes. Our gene of interest in pTRE was also digested with same enzymes and ligated with CMV IE promoter piece extracted from gel electrophoresis. Results are shown below (Figure 3)
Transfection
In order to show that the parts of the system work, after cloning we transfected the plasmids that can be clonned into eukaryotic cells. .In order to show that the system worked, we had planned to use the gastric cancer cell line, AGS. We obtained the cell line from another lab, but we had problems culturing them. . Therefore, for the time being, we performed our transfection experiments with HEK293-T cells. Below are the transfection conditions and microscope images from the transfection experiments we performed
In the images, DsRed protein production is seen. However, since the cell line we used is not a cancer cell line, the amount of protein produced is small.
Protein was isolated from the cells seen in the microscope images. Following isolation, we measured the DsRed florescence levels of the samples... Results are listed below
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 835
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 593
Illegal BamHI site found at 876 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 535
- 1000COMPATIBLE WITH RFC[1000]
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