Team:Worldshaper-Wuhan/solution-and-design

• Home
• About Us
• Background
• Project
  • Solution and Design
  • Results
  • Model
  • Parts
  • Measurement
  • Procedure
  • Notebook
• Safety
• Human Practice
• Collaboration
• InterLab
• Attributions
• Awards
  • Achievement
  • Survey Result
  • Applied Design
  • Silver
  • Gold

Menu

Street Address

City, State, Zip

Phone Number

Your Custom Text Here

• Home
• About
• Background
• Project
  • Solution and Design
  • Results
  • Model
  • Parts
  • Measurement
  • Procedure
  • Notebook
• Safety
• Human Practice
• Collaboration
• InterLab
• Attributions
• Awards
  • Achievement
  • Survey Result
  • Applied Design
  • Silver
  • Gold

Solution and design

 

 

Our project constructed a mir-21 sponge containing marker gene of GFP for the detection and inhibition mir-21 in cell lines, and offers a non-invasive and highly sensitive approach for early diagnosis and treatment of colorectal cancer in the future.

miRNA sponge, which contains multiple target sites complementary to a miRNA of interest, is used to be a inhibitor of miRNA. Here, we proposed miRNA sponge could be a monitor to detect the change of miRNA expression in cells. After transfection of the miR-21 sponge, sponge RNAs can attract miR-21, inhibiting the expression of eGFP reporter. The expression of miR-21 should be monitored by measuring the expression of eGFP reporter (Fig.1).

Fig.1. Model for the effect of miR-21 sponge in cells.

In the absence of sponge treatment, lots of miR-21 existed in cells. After introduction of the sponge transgene, sponge RNAs are expressed at a high level and attract miR-21, inhibiting the expression of eGFP reporter. The expression of miR-21 should be identified by their eGFP reporter expression.

Pervious studies indicated sponges containing bulged sites that are mispaired opposite miRNA are more effective (Ebert MS et al. Nat Methods. 2007 4:721-6.). Typical sponge constructs contain 2 to 10 binding sites separated by a few nucleotides each. Here, we designed two different mir-21 sponges with different binding sits to test the efficiency of sponges for detecting miRNA. One mir-21 sponge contains two miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-mir-21-sponge-2s). Another one mir-21 sponge contains six miR-21 binding sites with 3-nt spacers for bulged sites (pEGFP-mir-21-sponge-6s) (Fig.2).

Fig.2. The imperfect pairing between a miR-21 and a sponge with bulged binding sites is diagrammed. The miRNA seed region is showed by red.

In our research, pEGFP-C1 was used as the vector of our Biobrick device (Fig.3). pEGFP-C1 encodes a red-shifted variant of wild-type GFP, which has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) The recombinant EGFP vector can be transfected into mammalian cells using any standard transfection method. Mir-21 sponge contains miR-21 binding sites was inserted into the 3’UTR of a reporter gene encoding EGFP driven by the CMV promoter in the pEGFP-C1 plasmid.

Fig.3. The structure of pEGFP-C1 plasmid.

 

 

 

Wuhan, China .   New York, USA