Part:BBa_K1639010

mammalian LacI with miR-373 binding site

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-373 rate. In gastric epithelial cells level of this mirna is low but in gastric cancer cells 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.

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)
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

Cloning and Expression

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1170
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1193
  
• 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 1186