Part:BBa_K1940009

microRNA-34c detector 2.0-2-2

We designed this part to detect the abundance of microRNA-34c which is significantly related with Alzheimer's disease (AD). In device 2.0, we want to achieve the positive relationship of the fluorescent intensity and the concentration of microRNA-34c. We had tried two NOT logic gate, tetR-Ptet and lacI-Plac, with the composition of 4 different strength constitutive promoters (Figure 1. & Figure 2.). In BBa_K1940004, the promoter strength is 0.47(Figure 3.).

Figure 1. Device 2.0 construct design.

Figure 2. Promoter strength.

Figure 3. Device 2.0-2-2 construct design.

Experimental approach and results

We transformed well-constructed plasmid in to competent E. coli cell, then we got Device 2.0. Finally, we shifted microRNA in to Device 2.0 and did a continuing florescent intensity report (Figure 4a. & Figure 4b.).
It can be inferred that for each system, an observation of an increase in the expression of the florescence intensity is detected due to the addition of the microRNA. Therefore, a conclusion that the addition of the microRNA to the system repressed the expression of the repressor could be made. We could say that the expectation of our experiment has appeared.

Figure 4a.

Figure 4b.

Therefore, in contrast of repressors with different promoter strengths (Figure 5.), we minus florescence intensity expressed by each system after the microRNA add to them with the florescence intensity each system expressed before the microRNA add in to the system to get the Δ of florescence intensity of each system. According to the figure, we can tell that in the first place, each Δ of florescence intensity-time function figure of each system appeared to be a bell curve. This means that the window period of the system 2.0 still exists. Along the growth rate of the bacteria speed up, the division of the bacteria will make the florescence intensity decrease to the same level of the period when the microRNA hasn’t introduced to the system yet. Therefore, the Δ of florescence intensity could be zero. Moreover, when the promoter strength of the repressor protein increase, the max value of Δ expressed a gradually decrease tendency. This decrease is especially well expressed when we focus on the ‘Lacl-Plac NOT gate gene route’. Therefore, we can draw a conclusion that the different combination with different promoter strength could produce a theoretical ‘detectable minimum value ’. We could say that the concentration of the microRNA is below this theoretical value, predictable low florescence intensity would occur because of the low concentration of microRNA’s incapability of covering the amount of the repressor’s production. In another word, retroactivity would occur in our experiment. When the promoter strength of the repressor gradually decreases, the ‘detectable minimum value’ will decrease as well. This means the detectable range will increase. For microRNA with the same concentration, a combination with small promoter strength will output a relatively higher ‘maximum value of Δ (florescence intensity)’ which perfectly fit our observation result.

Figure 5.

To choose the most favorable repressor for Device 2.0, as the figure 5 present, Lacl-Plac route provide a distinguishable data under different promoter strength. This means when the promoter strength is settled, for different microRNA’s concentration, there will be a great possibility for Lacl-Plac to produce very distinguishable data that could be applied to real detection and experiment. On the other hand, for the NOT gate route consisted by TetR-Ptet, it didn’t express a very distinguishable difference of the maximum value of Δ (florescence intensity) when under the conditions with a clear difference in promoter strength. Still, we cannot say that the TetR-Ptet is not a favorable element for the construction of the system 2.0. Possible reason is that the system reached to the ‘detectable minimum value’ in advance due to the weak binding ability of the repressors to the DNA. Therefore, maximum promoter strength will produce a high Δ value. In this case, the system is favorable to work under a condition of a low concentration of microRNA. Moreover, we think theoretically should exist a route which repressor has strong binding ability to the DNA and favors the condition with a high concentration of microRNA.

Sequence and Features

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1367
  
• 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 2388