Team:MIT/miRNA

From 2014.igem.org

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Revision as of 02:49, 18 October 2014

Description
Outcome
Experiments

Low Sensor
High Sensor
L7ae
Single-Input
Multi-Input

Parts

miRNA Detector Module

sensing Alzheimer's through multi-input miRNA-based logic

Description

miRNAs (microRNAs) are short, noncoding strands of RNA that facilitate gene silencing - a single miRNA degrades an mRNA through a process involving complementary base-pairing between the miRNA and part of the mRNA sequence. A cell’s miRNA profile comprises the relative levels of all the miRNAs produced by that cell. Because miRNAs play a key role in regulating gene expression, it ought to be expected that a liver cell’s miRNA profile would differ significantly from that of a neuron. But more surprisingly, miRNA profiles can discriminate between identical cells in different conditions.

Neurons afflicted with Alzheimer’s disease display an miRNA profile significantly different from that of healthy neurons (A blood based 12-miRNA signature of Alzheimer disease patients, Leidinger et al, 2013). The miRNA subgroup aimed to use this difference as an approach to detecting Alzheimer’s disease. Our goal was to build a set of genetic sensors to specifically detect the miRNA profile of a neuron with Alzheimer’s and initiate a specific biological response upon doing so.

Our strategy took its inspiration from a similar detection circuit demonstrated to respond to cancer onset (Multi-input RNAi-based logic circuit for identification of specific cancer cells, Xie et al, 2011) Through existing research, we identified six miRNAs that are critically up- or down-regulated in Alzheimer’s neurons. Using the inverting logic inherent to miRNAs, we designed detection circuits to release a response factor upon sensing either heightened or lowered levels of their target miRNA, and customized each circuit to use one of the six miRNAs as its input. Using the principles of combinational logic, we can integrate the inputs from all six of our miRNA sensors, and actuate our response only when all six miRNAs meet their critical threshold concentrations. This ensures excellent specificity for our circuit.

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Outcome

The miRNA detection team built individual sensing constructs for each miRNA. We determined input-output relations for our sensors using flow cytometry and found that our sensors respond to miRNA levels by modulating the production of a fluorescent reporter, exactly as we had predicted.

Future work on our sensors will focus largely on implementation concerns - tuning as well as integration. Although we have shown that our sensors respond on a digital level, this does not accurately model the dynamic chemical conditions of the intracellular environment. We would thus like to refine our sensor control. In the ideal case, a small shift in a critical range of miRNA concentration will result in a large output signal, so that the treatment response is both specific and substantial.

We only tested binary combinatorial inputs for our sensors (one high and one low, or two of each). The ultimate goal is to use all six sensors in tandem with one another. When we use more sensors, we achieve greater precision, but as a tradeoff we gain more variables that require keeping track. There is also the complication that the various miRNAs are not biologically present at the same concentrations, meaning that each of our sensors must be individually tuned for optimal response to its own miRNA. Because all six of our sensors actuate the same response, we must also ensure that one sensor does not become overstimulated, activating our treatment even in the absence of input from the other sensors. These are all issues that can only be answered through extensive iterative testing.

The miRNA sensing team has established a conceptual grounding for a detection mechanism that responds to cellular conditions in the fashion of a true biological system. It is worthwhile to note that our strategy is not Alzheimer’s-specific, and can be implemented with any disease with a characteristic miRNA profile. This can be a novel approach for diseases with poorly understood etiologies, such as Parkinson’s (MicroRNA profiling of Parkinson's disease brains identifies early downregulation of miR-34b/c which modulate mitochondrial function, Minones-Moyano, 2011)

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Experiments

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Low Sensor Construction

By cloning an miRNA target site 3’ to a gene coding a reporter protein, we can easily create a sensor that produces reporter protein only when miRNA levels are low enough to permit translation. In our experiments, we used a fluorescent reporter as a placeholder for rtTA, which would activate our treatment circuit.

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High Sensor Construction

Because miRNAs naturally silence genes, for our high sensor design we cloned miRNA target sites to a repressor protein that would block transcription of response protein at the low sensor. We chose to use the L7ae/K-turn to eliminate the possibility of crosstalk with other cellular activities.

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Repression of L7ae

Before coming to any conclusions about the success of our constructs, we needed to make sure that the L7ae/k-turn system worked correctly. To do this we expressed k-turn:eGFP with and without the presence of constitutive L7ae. We used eBFP as our normalizing transfection marker.

In the absence of L7ae, eBFP and eGFP levels scale linearly with each other, as expected when co-expressing two constitutive fluorophores. However, upon the addition of L7ae, eGFP production is completely silenced, indicating proper function of the L7ae/k-turn system.

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Single-Input Sensor Testing

We tested the sensitivity of our sensor constructs individually for each of the six miRNA that are upregulated or downregulated in Alzheimer's neurons. Our experiments were conducted by transfecting HEK293 cells with our sensor constructs because they do not endogenously express any of the miRNA's that we sensed for, thus they provided the perfect low miRNA environment. In order to artificially create a high sensor environment, we co-transfected siRNA along with our sensors.

Low Sensors
A
B	C
Low Sensor miR-144 in the presence and absence of siRNA-144 in HEK293 cells. Using red as our transfection marker and blue as our output, we expected that the sensor, when exposed to the inhibiting siRNA, would show repressed eBFP2 output. However the cytometry results in figure (A) shows that there is no difference between the 'on' and 'off' state. This conclusion is reinforced by the microscopy in (B) and (C). On the left the sensor is in a low miRNA environment, and on the right the sensor is in a high miRNA environment.

A
B	C
Low Sensor miR-181c in the presence and absence of siRNA-181c in HEK293 cells. Using red as our transfection marker and blue as our output, we expected that the sensor, when exposed to the inhibiting siRNA, would show repressed eBFP2 output. However the cytometry results in figure (A) shows that there is no difference between the 'on' and 'off' state. This conclusion is reinforced by the microscopy in (B) and (C). On the left the sensor is in a low miRNA environment, and on the right the sensor is in a high miRNA environment.

High Sensors

A
B	C
High Sensor miR-30d in the presence and absence of siRNA-30d in HEK293 cells. Using red as our transfection marker and green as our output, we expected that the sensor, when exposed to the inhibiting siRNA, would show repressed eBFP2 output. However the cytometry results in figure (A) shows that there is no difference between the 'on' and 'off' state. This conclusion is reinforced by the microscopy in (B) and (C). On the left the sensor is in a low miRNA environment, and on the right the sensor is in a high miRNA environment.

A
B	C
High Sensor miR-125b in the presence and absence of siRNA-125b in HEK293 cells. Using red as our transfection marker and green as our output, we expected that the sensor, when exposed to the inhibiting siRNA, would show repressed eBFP2 output. The cytometry results in figure (A) shows that there is difference between the 'on' and 'off' state. This conclusion is reinforced by the microscopy in (B) and (C). On the left the sensor is in a low miRNA environment, and on the right the sensor is in a high miRNA environment.

A
B	C
High Sensor miR-146a in the presence and absence of siRNA-146ain HEK293 cells. Using red as our transfection marker and green as our output, we expected that the sensor, when exposed to the inhibiting siRNA, would show repressed eBFP2 output. However the cytometry results in figure (A) shows that there is no difference between the 'on' and 'off' state. This conclusion is reinforced by the microscopy in (B) and (C). On the left the sensor is in a low miRNA environment, and on the right the sensor is in a high miRNA environment.

A
B	C
High Sensor let-7f in the presence and absence of siRNA-7f in HEK293 cells. Using red as our transfection marker and green as our output, we expected that the sensor, when exposed to the inhibiting siRNA, would show repressed eBFP2 output. The cytometry results in figure (A) shows that there is no difference between the 'on' and 'off' state. This conclusion is reinforced by the microscopy in (B) and (C). On the left the sensor is in a low miRNA environment, and on the right the sensor is in a high miRNA environment.

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Multi-Input Sensor Testing

Our next goal was to test multiple sensors at once to ensure that they interacted with one another in the manner we desired. We did not test all six sensors together, as this would have required too much time. Instead we ran tests of various sensor pairs: two high sensors together, two low sensors together, and one high with one low sensor. Together, these encompass all the possible pairwise interactions in our detection module.

Two Low Sensors

Two High Sensors

One High and One Low Sensor

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Parts

pENTR_hEF1a
MAV1212-RFP
pENTR_L7ae
MAV1212_hEF1a_L7ae
MAV1212_hEF1a_eBFP2
Low sensor: eBFP2_miR-144
Low sensor: eBFP2_miR-181c
High sensor: L7ae_miR-30d
High sensor: L7ae_miR-146a
High sensor: L7ae_miR-125b
High sensor: L7ae_miR-let-7f
Low sensor: eBFP2_miR-144_miR-181c

Retrieved from "http://2014.igem.org/Team:MIT/miRNA"

@@ Line 119: / Line 119: @@
 <a name="4"></a><h3>Single-Input Sensor Testing</h3>
-We tested the sensitivity of our sensor constructs individually for each of the six miRNA that are upregulated or downregulated in Alzheimer neurons.
+We tested the sensitivity of our sensor constructs individually for each of the six miRNA that are upregulated or downregulated in Alzheimer's neurons. Our experiments were conducted by transfecting HEK293 cells with our sensor constructs because they do not endogenously express any of the miRNA's that we sensed for, thus they provided the perfect low miRNA environment. In order to artificially create a high sensor environment, we co-transfected siRNA along with our sensors.
-To test our high sensors, we ran the same experiment as before, but instead of using constitutive L7ae, we used our high sensor constructs, which produce L7ae at a level dependent on the concentration of miRNA being sensed. We used custom-designed siRNAs to control the level of tinput for the sensors.
-To test the low sensors, we expressed only the low sensor constructs, and again used siRNAs to modulate our input.
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