Team:ZJUT-China/Proof Of Concept
The feasibility of gene circuits has been demonstrated on the engineering success page. Therefore, we will elaborate on how to prove the feasibility of our entire project concept on the proof of concept page.
We, the iGEM team ZJUT-CHINA, developed a Cell-Free RNA biosensor to detect some specific RNA biomarkers, based on a special gRNA (igRNA) from the CRIPSR/Cas9 system. To prove our concept, we tested every part of the biosensor, which can be divided into three principal components: a bio-receptor with a recognition element, a transducer component and a detection system (Figure 1).
Figure 1. Schematic of the concept of the biosensor.
In the project, the CXCL9 mRNA[1-2] was chosen as the bio-receptor, and CRISPR/cas9 system was chosen as the recognition element. We designed gRNA to become 'igRNA'[3], which was capable of hybridizing with CXCL9 mRNA (Figure 2).
Figure 2. Schematic of the CXCL9 mRNA, igRNA and the hybridization of CXCL9 mRNA-igRNA Complex.
Before the proof of concept experiments, we used NUPACK webserver to analyze the 2D structure of it, ensuring the hybridization of CXCL9 mRNA and igRNA (Figure 3).
Figure 3. MFE structure of CXCL9 mRNA (a), igRNA (b), CXCL9 mRNA-igRNA (c).
We successfully proved that igRNA was able to hybridize with CXCL9 mRNA through experiments, like EMSA, cleavage with Cas9 protein in vitro and so on.
Our transducer component is the Cell-Free system carrying a cascade of gene circuits.
Through the cascade of gene circuits, the biosensor will not produce green fluorescence when RNA biomarker is absent, while in the presence of RNA biomarker, Cas9 can cleave the tetR gene to produce green fluorescence (Figure 4).
Figure 4. Schematic of the concept of transducer.
The proof experiments were conducted in the Cell-Free system, we verified the function of every part of the gene circuits and the whole transducer (Figure 5).
| Group1 | Group2 | Group3 | Group4 | Group5 | |
| dCas9 | + | + | + | + | + |
| +C gRNA | + | - | - | - | - |
| -C gRNA | - | + | - | - | - |
| igRNA | - | - | + | + | + |
| CXCL9 | - | - | - | + | - |
| non-mRNA | - | - | - | - | + |
Figure 5. Cell-Free testing of CXCL9 mRNA-igRNA complex with the Cas9 targeting the tet repressor releases the expression of deGFP. Bar graphs show the average fluorescence intensity emitted by the fluorescent reporter construct.
The endpoint measurement of cascade with CRISPR/Cas9 system shows that CXCL9 mRNA-igRNA complex binding with Cas9 protein makes the fluorescence intensity (Group 4) much higher than the negative control (Group 5), which indicates that the CRISPR/Cas9 system releases the inhibition of tetR to deGFP when the biosensor detects the RNA biomarker (CXCL9 mRNA). So our biosensor is able to detect RNA biomarkers successfully.
To broaden application scenarios for the biosensor, we designed hardware to detect the fluorescence intensity, which consists of an illuminator for fluorescence excitation and a fluorescence detector (Figure 6).
Figure 6. Design of the handheld illuminator. The illuminator provides easy visualization of the sensor’s fluorescence output.
(A) 3D rendering of the illuminator.
(B) The internal structure of the illuminator. Back case includes LEDs for fluorescence excitation (Top). Front case has a window fitted with a light filter (Bottom).
(C) The lighting when turning the LEDs on without front case.
(D) Visualization of different concentrations of deGFP protein by illuminator.
After every part was tested, the hardware was utilized to test our biosensor (Figure 7). The group 4 from "the verification of cascade with CRISPR/Cas9 system" was expressed in the harware to be measured.
Figure 7. Diagram of the fluorescence intensity under different reaction time.
(A) 12 μL Cell-Free reagent carrying the plasmid of cascade with CRISPR/Cas9 the system, incubating at 29℃. In every diagram, the left is negative control and the right is test group.
(B) Endpoint measurements for the verification of the biosensor with the hardware.
The endpoint measurement of cascade with CRISPR/Cas9 system shows that test group makes the fluorescence intensity much higher than the negative control, which indicates that the CRISPR/Cas9 system releases the inhibition of tetR to deGFP when the biosensor detects the RNA biomarker (CXCL9 mRNA).
So our biosensor is able to detect RNA biomarkers successfully.
RNA biomarkers can be detected by our Cell-Free biosensor. The measurements, experiments, and hardware testing demonstrated that without RNA biomarker, the fluorescence intensity is significantly lower than with the RNA biomarker that is present. As a result, we have demonstrated the feasibility of our concept. Moreover, the RNA biomarker concentration is related to the sensitivity of the biosensor, but we do not have much more time to test this attribute, so we put forward a plan for the next step.
1. Sensitivity of Biosensor: CXCL9 mRNA with different concentration gradients is set for quantitative detection of fluorescence intensity and device inspection to determine the minimum concentration that can be detected.
2. Universality of Biosensor: Multiple igRNAs and corresponding RNA biomarkers were designed for testing.
3. Convenience of Biosensor: Currently, Cell-Free system reagents need to be stored in a -80℃ refrigerator. For readily application of the device in areas without experimental conditions, we will provide freeze-dried reagents.
1. Compatibility of illuminator: The illuminator's test tube holder holds only 200 μL test tubes. Different sizes of test tube holders are required to accommodate different volumes of test tubes.
2. Sensitivity and accuracy of fluorometer: The minimum detection limit and accuracy of the fluorometer need to be improved further with more tests.
3. Power supply of fluorometer: Currently, fluorometers require an external power source, which limits their application. A battery-powered fluorometer is suitable for a broader range of scenarios.
1. Strengthen follow-up communication with kidney transplant patients, carry out large-scale social research, collect patients' inner needs and the public's suggestions for our device.
2. Contact Environmental Protection Agency (EPA) to address medical waste recycling issues.
3. Contact software experts to improve the construction of the RNA biomarkers database.
4. Communicate with medical instrument companies to find out the deficiencies of the device and prepare for the device launching on the market.
1. Modeling was used to simulate the biomarker of RNA with different concentrations, which was combined with subsequent experiments in the wet group and provided guidance.
2. MGapt part was used to test various parameters related to modeling, especially related parameters of the promoter P32, which was the strongest promoter in Cell-Free system except for P70a and P28 promoters.