Regulatory
mLacI

Part:BBa_K1639011

Designed by: Nurgeldi Bazarov   Group: iGEM15_ATOMS-Turkiye   (2015-09-15)

mammalian LacI with miR-223 and miR-21 binding sites

In our project second step of cancer module is based on production of LacI so that it will suppress expression of DsRed. Binding site on 3' end of LacI makes it regulatable and limits it's expression only in cells with low miR-223 and miR-21 levels. miR-21 has high rate of production in most cancer types. In gastric epithelial cells level of this miRNAs are low but in gastric cancer cells their level is very high.


Usage and Biology

"m" in mLacI stands for mammalian, this part is mammalian codon optimized of bacterial LacI and also it contains Nuclear Localization Signal to show its activity on lac operators.

Figure 1
Research published by Zhen Xie, et al. in 2011 is central in cancer module of project. According to this research whole system composed of 5 different miRNas can classify cells based on intracellular miRNA levels. The system depends on repression of flourescent protein production.(Figure 1) With all inputs included, the response function is well approximated by a Boolean expression

miR‐373 AND miR‐21‐223

AND NOT(miR‐375)

AND NOT[miR‐26a]
To achieve this binding sites complementary to high miRNas (cancer cells) were put on mLacI and tTA activator of pTRE-mLacI procuing vector. On the other hand low-miRNA binding sites were added to DsRed mRNA. As the level of these miRNas would be different in normal cells, flourescent protein would be repressed by high-miRNas in normal cells but actually are at low levels in cancer cells and repressor mLacI wouldn't be inhibited by low-miRNas in normal cells but actually are at high levels in cancerous tissue and likewisely repression of activator wouldn't be maintained because of same reason. More simplified expression is in diagram (Figure 2)

Figure 2:Schematic explanation of whole system. Low miRNas bind directly to flourescent proteins.High miRNas bind to repressor of DsRed and to actvator of repressor

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

ATOMS-Turkiye cancersw 8.png
ATOMS-Turkiye cancersw 9.png

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
ATOMS-Turkiye cancersw 10.png
ATOMS-Turkiye cancersw 11.png

ATOMS-Turkiye cancersw 12.png
ATOMS-Turkiye cancersw 13.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1172
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1201
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 562
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 787
    Illegal BsaI.rc site found at 1165


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