Part:BBa_K3179107

LSBR5n3

LSBR5n3

Based on our analysis, we designed three different miRNA targets. We inserted it into the following miRNA detection plasmid LSBr5and3 (Figure 1a) to verify its independent ability to work and detect the expression of the corresponding miRNA in different HEK293. To verify whether the system causes tandem, we constructed three different plasmid contains 3 miRNA targets independently (miR-592, miR-663b and miR885-5p) were tested under different miRNA induction. After the plasmid is transcribed into the cell, the endogenous miRNA binds to the target on the mKate mRNA, blocking the expression of mKate, which cause a change in the value of mKate/EBFP that can be read by flow cytometry. The detection plasmids were tested under different miRNA induction.
By this method, we can compare the expression of the endogenous corresponding miRNA with the Positive Control which does not carry the miRNA target. At the same time, we transferred miR-592 mimetic, miR-885-5p and miR-663b inhibitors into corresponding cells to detect the regulation of the corresponding miRNA target by miR concentration, thus verifying the rationality and feasibility of single miRNA target design.
In the flow cytometry analysis (Fig. 1b, 2), we refer to the group with the lower affected group of mKate as the positive group, and the group with the larger influence of mKate as the negative group. We can identify a group of cells that are not obvious in the negative group (hexagonal circled part) in the positive group, and there is a significant difference in the ratio of cells between the positive group and the negative group in the interval of mKate/EGFP<0.4. Thus, we believe that miRNA targets can respond to changes in miRNA concentration.

Figure 1:Flow cytometry of miRNA detection plasmids

a. The detection plasmid is mainly composed of two fluorescent expression systems. The target of the target miRNA target is inserted into the 3'UTR sequence of mKate. If the miRNA target can work normally, the fluorescence ratio of mKate/EBFP can be changed. b. In the results, the positive group has a common cell population, which shows that all three miRNA targets work properly. At the same time, the concentration of endogenous miRNA corresponding to HEK293 can be displayed.

Figure 2: Density analysis of positive and unique cell populations revealed that the positive group had a deviation from the negative expression of mKate in the negative group

Usage and Biology

This part is a part of measurement, which consists of mKate, miRNA target and EBFP and is put in the vector eEF1α. This plasmid has two eEF1α promoter, one promoter the transcribe of miRNA target and mKate, another promote the transcribe of EBFP. Because the efficiency of two promoter are similar, the transcription of mKate and EBFP’s mRNA are totally same. So, we can calculate the transcription by the expression of EBFP. Meanwhile, the mRNA of mKate has the miRNA target, so, we can get the amount of free miRNA by the ratio between EBFP and mKate which is also called the effective amount. In our design, this part is used to calculate the effective amount of three kinds of miRNA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 83
  Illegal AgeI site found at 1547
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1376
  Illegal BsaI.rc site found at 2430
  Illegal SapI.rc site found at 758