Results

Results

PART 1 | in silico Design & Estimation

PART 2 | Binding pattern analysis and visualization

Data deleted after the judging session

These images are the results of the Protein-Aptamer Docking(PAD) experiment. To use aptamers in LFA, they must be bound vertically with protein due to steric hindrance [1]. From PAD images, we confirmed that N56 aptamer binds perpendicularly to the CEA.

As a result, the visualization of the nearly perpendicular coupling between the CEA-N56 pair demonstrates its suitability for LFA sensors, which has not been reported in previous papers. We report this for the first time in iGEM.

PART 3 | CFE & EMSA

1.CFE (Cell-Free-Expression)

One of our goals in synthetic biology is the production of proteins through a Cell-Free Expression system (CFE). CFE is anticipated to emerge as a potent tool in the future of synthetic biology. Therefore, we executed a Design-Build-Test-Learn (DBTL) cycle to refine the environmental conditions of the protein expression process. During the initial CFE cycle, we encountered challenges in the overexpression of CEA proteins. Subsequently, we conducted troubleshooting to solve the underlying issues in the experimental conditions and proceeded with a second cycle of CFE.

2.EMSA(Electrophoretic Mobility Shift Assay)

The EMSA serves as a powerful tool for confirming the in vitro binding between specific nucleic acids and proteins. In our study, we aimed to demonstrate the interaction between CEA and the N56 aptamer using this technique. The observation of band-shift in the mobility of the FAM-tagged aptamer, when compared to controls, provides evidence of the binding event.

The Electrophoretic Mobility Shift Assay (EMSA) is a valuable tool for confirming in vitro interactions between specific nucleic acids and proteins. Our study aimed to use this technique to demonstrate the interaction between CEA and the N56 aptamer. This demonstration is usually achieved by observing a band shift in the mobility of the FAM-tagged aptamer when compared to control samples.

Regrettably, due to constraints, including tight deadlines, we were unable to carry out the EMSA as planned. Nevertheless, KUAS remains committed to advancing our experiments. We are determined to incorporate any relevant findings from the EMSA experiment into an upcoming presentation video as soon as circumstances permit.

Despite the absence of an EMSA experiment, we are confident in the binding interaction between the N56 aptamer and CEA. This confidence is grounded in prior research and the results from our modeling and PAD experiments.

Discussion

PART 1 | Advantages & Significance

Here are the advantages and significance of this research.

Firstly, We designed CEA N domain DNA templates(parts) for in vitro expression(CFE). There are only a few DNA template parts for CFE, it would be a great milestone for future iGEM projects.

Secondly, uploading aptamers to the parts registry along with corresponding documents contributes to the accessibility and dissemination of iGEM projects.

Additionally, proposing a new paper-based diagnostic device signifies progress in point-of-care technology, improving the practicality of medical diagnostics. The revealed analysis of the CEA-N56 binding complex provides insights into potential biosensor applications for the cervical cancer biomarker CEA. The observed near-vertical binding pattern highlights the potential for sensor development, going beyond the identification of specific aptamers.

PART 2 | Further Research Suggestions

In our future research, we will focus on advancing the "CEApture". As we proved the aptamer N56 and the biomarker CEA are suitable for quick diagnosis called Lateral Flow assays(LFA), quantifying how well these elements bind together is needed. The dissociation constant, Kd, might be measured by a powerful method called Confocal Laser Scanning Microscopy(CLSM).

To make it commercialized in the real world, our team plans to design detailed LFA sensors, especially focusing on color-changing methods/two-line indications on the pads. The fact that we've already proven gives us enough confidence that "CEApture" will work well in real life. We hope that it will help more people accept and use LFA technology for cancer diagnosis in their lives.

In conclusion, our experimental findings proved the clinical viability of the CEA protein and N56 aptamer pairing for application in an LFA sensor. This establishes a scientific foundation for the development of the LFA sensor-based pad that can be used as cervical cancer diagnostic device, termed 'CEApture.'
  

[1] J.-G. Walter, Ö. Kökpinar, K. Friehs, F. Stahl, and T. Scheper, “Systematic Investigation of Optimal Aptamer Immobilization for Protein−Microarray Applications,” Analytical Chemistry, vol. 80, no. 19, pp. 7372–7378, Aug. 2008, doi: https://doi.org/10.1021/ac801081v.