Part:BBa_K1940001

microRNA-34c detector 1.0

We designed this part to detect the abundance of microRNA-34c. Anti-microRNA-34c (BBa_K1940000) was insert after a constitutive promoter (BBa_J23106) which express GFP. As anti- microRNA-34c is sensitive to the expression levels of miR-34c, we would have a direct impact on GFP’s expression. Determining GFP’s fluorescence intensity differences will justify the content amount of microRNA-34c.

Figure 1: Construct design.

Experimental approach

For testing this device, we transform the constructed plasmid in to E.coli competent cell. Shift microRNA-34c in to our system, continuous observation on fluorescence intensity’s change.

Proof of function

According to the figure(Fig.2), between the time intervals of 12-19 Hrs, subject system (transforming microRNA) result in significant lower fluorescence intensity than comparison group.

Figure 2

Therefore, we start to test the different microRNA’s concentration’s impact. We separately add 10uM unrelated microRNA, 10uM target microRNA and 20uM target microRNA in to our system. Afterword, use Flow cytometry to collect data at 10 Hrs (Fig.3). Needless to say, average expression rate shows a fine deceasing gradient graphic. Indicated that our system could not only determine the existence of microRNA but also the quantity of microRNA as well.

Figure 3

Sequence and Features

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Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 186
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 50
  Illegal BsaI.rc site found at 183
  Illegal BsaI.rc site found at 864