Part:BBa_K1940000
anti-microRNA-34c
Anti- microRNA-34c can be used as a miR-34c detector. MiR-34c is significantly upregulated in Alzheimer's disease (AD) patients. Several researches have suggested that strategies to manipulate miR-34c could open a suitable novel virus-free therapeutic avenue to treat cognitive diseases. In this part, we designed 4 series antisense strand of miR-34c which are linked by 4 linkers. Anti- microRNA-34c is sensitive to the expression levels of miR-34c. And combined with a reporter, we can diagnose AD accurately and rapidly.
Proof of function
When we examine the effect of anti-miRNA insertions on GFP expression in the initial state(Fig.1-1--4 ), we obtained the following data(Fig.1-5).
From the figure, both “promoter-antimicro-RBS” and “RBS-antimicro-GFP” could express GFP regularly, but the fluorescence intensity of “promoter-antimicro-RBS” increased significantly. We hypothesize that perhaps the sequence of anti-miRNA itself can act as a “strong promoter” or “strong RBS”, so as to increase the expression quantity of GFP. Hence, we constructed additional gene circuits(Fig.2) to prove our ideas.
Also, we conducted fluorescence detection and obtained the following results (Fig.3 & Fig.4 ).
It can be seen that the express ability of the system lose when promoter or RBS is removed from the expression. This suggests that the anti-miRNA sequence itself cannot act as an independent element to initiate transcription or translation of gene.
Therefore, the increase in GFP expression of promoter-antimicro-RBS could be explained by the interaction between anti-miRNA sequence and promoter or RBS, which indicates that there are certain problems about the insulation between components, which is caused by the design itself. We hope that the future of the team can develop a better solution to this problem.
Next, we used a flow cytometry to collect data for more detailed view of the expression of the promoter-antimicro-RBS device. Flow cytometry is method of measuring the size, density, fluorescence type, and fluorescence intensity of a particle (single bacterial) level. We performed the same flow cytometry experiments on the same samples as Figure.4. The results are demonstrated below (Fig.5 & Fig.6). We observed a very interesting phenomenon from the data of the flow cytometry: Comparing to Fig. 6, it can be seen that the expression of GFP in the “promoter-antimicro-RBS” was consistent with that in the “normal expression” between 2 and 5 hours, and the results of the flow cytometry showed good simple peak results (Fig.6).After 6 hours, the GFP expression of the “promoter-antimicro-RBS” in the microplate reader was gradually higher than that in the “Normal expression”. At first, we think that the different growth conditions of bacteria may lead to this difference, but the data of flow cytometry bring different interpretations: First, from the comparison of the total profiles, we can see that the two devices have no significant difference in growth rate ( this can also be demonstrated by the number of particles per volume of flow cytometer, this data is not given), but for the distribution of fluorescence intensity, “promoter-antimicro-RBS” changed significantly after 7 hours. The bacterial population differentiate, and concentrate in two regions. The single peak pattern in the data gradually become double peak (Fig.6). We do not know why the bacterial population spontaneously generates and differentiation, and we hope that the future team will be able to explain this issue a reasonable explanation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 143
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 7
Illegal BsaI.rc site found at 140
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