E N G I N E E R I N G
Module I: Verification of Aptamer Binding Ability
Cycle 1 - Verification of reported thrombin aptamer sequence
Design
Aptamers are short oligonucleotide sequences (either DNA or RNA) typically derived from nucleic acid libraries using an in vitro selection technique known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). These oligonucleotide fragments can bind to various target molecules with high specificity and selectivity, making them widely utilized in biosensors. According to previous studies, there are three well-studied aptamers with strong affinity for thrombin, measuring 15 nt, 29 nt and 40 nt in length, respectively. Therefore, we decided to test the affinity of these three aptamers to thrombin and choose two as the foundation for subsequent experiments. There are several methods to assess the binding strength of aptamers to thrombin, among which Electrophoretic Mobility Shift Assay (EMSA) and Surface Plasmon Resonance (SPR) are two commonly used and sensitive techniques. Consequently, we employed these two methods to validate the affinity of the aptamers for thrombin.
We aim to use EMSA to validate the affinity of three different aptamers for thrombin (Figure 1). To detect the single-stranded aptamer after electrophoresis, we added a random extension (Figure 2) to form partial double-stranded structure, so that GelRed incorporation could be enhanced.
Figure 1: Principle of Electrophoretic Mobility Shift Assay (EMSA). In this assay, aptamers are incubated with thrombin or left unbound, followed by non-denaturing polyacrylamide gel electrophoresis to separate the components of the system. The appearance of free bands indicates aptamers that do not bind to thrombin, whereas shifted bands reveal aptamers that successfully bind to thrombin.
Figure 2: The design of aptamer-linker-probe complex. This structure enlarged the DNA molecule while its binding activity was not weakened.
EMSA provided a relatively rudimentary validation of the binding interactions. To achieve a more quantitative and precise characterization of the interactions between the 29-mer/40-mer aptamers and thrombin, we employed Surface Plasmon Resonance (SPR) for testing (Figure 3).
Figure 3: Schematic of the Surface Plasmon Resonance (SPR) Experiment. Biotinylated aptamers are immobilized on the SPR chip via streptavidin (SA) binding. Thrombin is then introduced to flow over the chip. When binding occurs, it causes a change in the resonance angle, indicating an interaction between the thrombin and the aptamers.
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The three aptamers were synthesized by Ruibo company, while thrombin was procured online. To immobilize the aptamers on the chip to perform SPR, we synthesized two biotinylated aptamers through a commercial provider. Subsequently, experiments were conducted using a SPR instrument.
Test
For EMSA, after thrombin and aptamers were diluted with the proper buffer, reaction systems were built with three different aptamers. 15-mer, 29-mer and 40-mer aptamers were tested, and a gradient concentration of thrombin were applied to reflect the binding affinity. After the aptamers were co-incubated with thrombin for 60 minutes, a 12% non-denaturing polyacrylamide gel electrophoresis was performed. The gel was then stained by fluorescent dye GelRed. The results are shown below.
Figure 4: Native-PAGE results of EMSA. Of the two figures, Lane 1, marker; Lane 2, control group with 15-mer, 29-mer and 40-mer aptamers and NO thrombin. All aptamers were at a concentration of 10 pM. Figure 4-2A Lane 3-5, 0.45 pM thrombin incubated with respectively 29-mer+40-mer, 15-mer+40-mer, 15-mer+29-mer; lane 6-8, 15-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM. Figure 4-2B Lane 3-5, 29-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM.; lane 6-8, 40-mer aptamer incubated with gradient thrombin concentration of 0.45, 0.9 and 1.8 pM.
After immobilization of the aptamers, gradient-diluted thrombin was loaded to obtain the corresponding curves within the SPR buffer. Partial experimental results were fitted with a 1:1 binding kinetic model in order to calculate dissociation constant (KD).
Figure 5: Surface Plasmon Resonance (SPR) results of aptamer-thrombin binding. The sensorgrams illustrate the binding interactions between the 29-mer/40-mer aptamers and thrombin, showing real-time changes in refractive index. Curves represent the association and dissociation phases, providing insights into the binding kinetics and the affinity of the aptamers for thrombin. A & C: Thrombin was subjected to binding and dissociation tests by flowing gradient-diluted samples over the chip. B & D: Partial experimental results were fitted with a 1:1 binding kinetic model in order to calculate dissociation constant (KD).
| Aptamer | General Kinetics model | Quality Kinetics Chi² (RU²) | 1:1 binding ka (1/Ms) | kd (1/s) | KD (M) | tc |
|---|---|---|---|---|---|---|
| 29 | 1:1 binding | 1.03e0 | 2.14e+5 | 1.74e-4 | 8.16e-10 | 4.81e+7 |
| 40 | 1:1 binding | 4.58e+1 | 1.59e+5 | 1.85e-1 | 1.17e-6 | 5.29e+7 |
Table 1 Parameters fitted from 1:1 binding kinetic model. RU: resonance units; ka: association rate constant (M-1s-1); kd: dissociation rate constant (s-1); KD: equilibrium dissociation constant (M); tc: flow rate-independent component of the mass transfer constant.
Learn
The EMSA results indicate that all the three aptamers have strong binding affinity for thrombin, and 40-mer aptamer was suspected to form multimers, causing a strong band at the sampling hole and unclear bands at the target sites. After quantitative validation by SPR experiments, aptamer 29-mer demonstrated a strong affinity for thrombin, with a dissociation constant (KD) of 0.816 nM, while aptamer 40-mer had a much lower affinity, with a KD of 1170 nM. By accurately determining the association and dissociation constants, we have significantly bolstered our confidence in the results obtained from EMSA, laying a solid groundwork for the foundational concepts necessary for our subsequent system development.
Cycle 2 - Verification of MAGA-generated Aptamer Sequence
Design
Accurate and timely detection of disease biomarkers is crucial for effective diagnosis and treatment across various medical conditions. Traditional methods of aptamer screening, such as SELEX, are often slow, costly, and lack the precision needed for diverse applications. To overcome these limitations, we introduce MAGA: Make Aptamer Generally Applied—a universal machine learning-based platform designed to predict aptamer sequences that can target a wide range of disease biomarkers with high specificity and affinity. You may visit the model and the software pages for more information about MAGA.
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We conducted validation experiments using three aptamers predicted by MAGA, along with a randomly generated sequence as a control. The sequences were synthesized by a commercial company. Unfortuneately, we failed to conduct SPR experiments before the end of wet lab due to time and instrument limitations.
Test
Our predictive model not only identified aptamer sequences that showed high binding affinity to thrombin, but the structural predictions made using AlphaFold3 were also in excellent agreement with experimental data. We employed Electrophoretic Mobility Shift Assay (EMSA) to further validate that the predicted aptamer can indeed bind to the target protein. Although the model we designed predicts the sequences of RNA aptamers, we utilized DNA aptamers in our experiments.
Figure 6: Native-PAGE results of EMSA. All aptamers were at a concentration of 20 pM. MAGA_A, MAGA_C and MAGA_E are aptamers obtained from MAGA, and MAGA_D is randomly generated.
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It can be observed that the MAGA_A aptamer exhibits a shift band at elevated thrombin concentrations, indicating that our predicted aptamer sequence is likely capable of binding to thrombin. The successful application of the MAGA system to thrombin not only validates our approach but also sets the stage for its expansion to other disease biomarkers. This positions MAGA as a powerful, universal tool for aptamer screening, offering a blend of computational efficiency and experimental precision that could revolutionize the field of diagnostic medicine.
Module II: The Expression and Testing of the Protease-based (SPOC) System
Figure 7: Overview of protein purification work flow. The protein purification process begins with transformation (1), where the desired gene is introduced into a host cell. This is followed by selection (2) of successfully transformed cells on a selective medium. Next, protein production (3) is induced in the selected cells, which are then subjected to cell lysis (4) via ultrasonication to release the proteins. The lysate is then subjected to protein purification (5) using an AKTA system or gravity column. The purified proteins are analyzed (6) to assess the success of the purification process, and finally, an activity assay (7) is performed to evaluate the functionality of the purified proteins.
Cycle 1 - Protein Expression and Solubility Testing
Design
Our goal is to extract active cleavage proteases and conjugate them with aptamers that recognize disease biomarkers for diagnostic purposes. To achieve this, after investigation and discussions, we constructed plasmids to express full-length TEV and PPV proteases and their substrates, as well as two split-PPV proteases. These plasmids were introduced into BL21(DE3) strains for protein expression. The cells were then centrifuged and ultrasonically disrupted for subsequent solubility testing.
Figure 8: Plasmid profiles of TEVp-pET28a, PPVp-pET28a, FKBP_cPPVp-pET28a, FRB_nPPVp-pET28a, and GFP_truncated_linker(PPV)_YFP_truncated-pET28a.
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We outsourced the complete synthesis of all constructed plasmids to a company. The synthesized plasmids were then transformed into E. coli, where target protein expression was induced, followed by solubility verification.
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We conducted SDS-PAGE analysis on the supernatant and resuspended pellet from the sonicated cell lysates and found that all expressed proteins were located in the pellet, indicating that these proteins were insoluble and existed as inclusion bodies.
Figure 9: Solubility analysis of PPV, its split forms, and substrate. SU: supernatant; PU: precipitate. (A) Solubility analysis of FKBP-cPPV (27.3 kDa) and FRB-nPPV (24.9 kDa). Both proteins are mostly in PU, indicating insolubility. The EGFP control appears mainly in SU. (B) Solubility analysis of PPVp (27.7 kDa) and G-PPVs-Y (22.3 kDa). Both proteins are insoluble.
Unlabeled bands are due to sample loading errors.
Figure 10: SDS-PAGE analysis of protein solubility of TEV. AU: after ultrasonication; SU: supernatant after ultrasonication; FS: final supernatant. Lane1, AU 1-1, i.e., total proteins of bacteria after ultrasonication; Lane2, SU1-1, i.e., proteins in the supernatant after ultrasonication; Lane3, FS1-1, i.e., proteins in the final supernatant. MW of TEV is around 31.0 KDa. This result demonstrated that bacteria expressed TEV successfully. TEV is insolube as there is no corresponding band in supernatant after ultrasonication.
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As our target proteins are insoluble and form inclusion bodies, further extraction is required to solubilize them for enzyme activity assays.
Cycle 2 - Protein Extraction, On-column Refolding and Purification
Design
After confirming that TEV, PPV, and their substrates (including split-PPV) are present in inclusion bodies, we developed a denaturation-renaturation extraction method followed by nickel column purification to solubilize and purify our target proteins.
Figure 11: Workflow for extracting and purifying target proteins from inclusion bodies. AU: after ultrasonication (total proteins). SU: supernatant after ultrasonication. PU: precipitate after ultrasonication. SW: supernatant after washing. PW: precipitate after washing. PD: precipitate after dissolution. FS: final supernatant.
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We utilized an on-column refolding strategy combined with affinity chromatography to purify our target proteins from inclusion bodies. Initially, the inclusion bodies were solubilized using high-concentration urea solutions, which denature the proteins and release them from the aggregated state. For refolding, we applied a gradient of urea-NiA buffer on a nickel affinity chromatography column. This approach allows gradual removal of the denaturant while the protein is bound to the column, facilitating proper refolding into its native conformation. The purified proteins were then eluted using imidazole-containing buffers. Both the ÄKTA system and gravity chromatography were employed for this process, depending on the scale and requirements of each experiment.
See more details in our Protocol.
Test
We confirmed the identity and purity of the extracted proteins through SDS-PAGE and verified their functionality using enzyme activity assays. The SDS-PAGE analysis showed clear bands corresponding to the expected molecular weights of cPPV, nPPV, PPV, G-PPVcutsite-Y, and TEV, indicating successful extraction and purification.
Purification of PPV, split-PPV and its substrate:
Figure 12: Elution profile of the nickel affinity chromatography of cPPV, nPPV, PPV, G-PPVcutsite-Y without solubility tags. Method used for dissolving those proteins from inclusion bodies is on-column refolding. Blue line stands for UV curve and Red line stands for conductivity curve. Figure 12-A, elution profile of cPPV. Peak A stands for transmission peak, peak B stands for elution peak of cPPV. Figure 12-B, elution profile of nPPV. Peak A stands for transmission peak, peak B stands for elution peak of nPPV. Figure 12-C, elution profile of PPV. Peak A stands for transmission peak, peak B stands for elution peak of PPV. Figure 12-D, elution profile of G-PPVcutsite-Y. Peak A stands for transmission peak, peak B stands for elution peak of G-PPVcutsite-Y.
Figure 13: SDS-PAGE analysis of the cell lysate, the transmission peak and the elution peak after nickel affinity chromatography through AKTA or gravity chromatography. Those proteins were expressed without solubility tags. Figure 13-A, Lane2-4, total proteins of bacteria expressing cPPV, elution peak, transmission peak of cPPV; Lane5-7, total proteins of bacteria expressing nPPV; Lane9-10, elution peak and transmission peak of nPPV. All the proteins are purified through AKTA. Figure 13-B, Lane2, total proteins of bacteria expressing G-PPVcutsite-Y; Lane3, elution peak of G-PPVcutsite-Y through AKTA; Lane4, total proteins of bacteria expressing PPV; Lane5, elution peak of PPV through AKTA; Lane6, elution peak of G-PPVcutsite-Y through gravity chromatography; Lane7, elution peak of PPV through gravity chromatography. PPV wasn’t purified successfully in this picture. Figure 13-C, Lane2-3, elution peak of PPV through gravity chromatography; Lane4-5, elution peak of PPV through AKTA after ultrafiltration inside the ultrafiltration membrane; Lane6-7, elution peak of PPV through AKTA after ultrafiltration outside the ultrafiltration membrane. Lane4-5&Lane6-7 showed that the ultrafiltration membrane was integrated and leak of proteins did not happen.
Purification of TEV:
Figure 14: SDS-PAGE analysis of the elution peak after gravity chromatography. Lane1-3, elution peak after gravity chromatography. The results demonstrated that TEV was successfully purified.
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After successfully solubilizing and purifying our proteins from inclusion bodies, the next step is to verify their activity.
Cycle 3 - Protein Activity Assay
Design
Verifying the activity of the target protease is crucial to ensure that the enzymes used are functional. We designed an experiment to assess enzyme activity under controlled conditions. This involved testing two intact proteases, TEV and PPV, as well as one split protease. Detailed protocols for these experiments are available in our documentation.
Figure 15: Principle of protein activity assay. Substrates are incubated with proteases (PPV/TEV). Samples are extracted at different time points and subjected to denaturing polyacrylamide gel electrophoresis. The largest mass band represents the uncut substrate, while the two smaller mass bands indicate the cleaved products.
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For intact TEV and PPV proteases, we mixed precise amounts of enzyme with their substrates and added the appropriate amount of reaction buffer. The mixture was incubated at 30°C, and samples were collected at various time points. Reactions were halted using SDS loading buffer, and enzyme activity was confirmed by SDS-Page analysis, showing substrate reduction and cleavage product formation. For the split PPV protease, we included a fixed amount of rapamycin to induce dimerization and activation, which was verified through similar analysis.
See more details in our Protocol.
Test
We validated the activity of the extracted protease using SDS-PAGE analysis. Enzyme activity assays showed that full TEV protease extracted from inclusion bodies was active and could effectively cleave its substrates. Both full-length PPVp and split PPVp exhibited protease activity, but the split PPVp showed significant cleavage only after the addition of the inducer rapamycin, demonstrating the necessity of rapamycin for optimal activity and validating the feasibility of our SPOC system.
Protein Activity Assay of TEV:
Figure 16: Enzymatic activity assay of TEV protease extracted from inclusion bodies.MW: MBP-tevS-G-Y: 65.6kDa G-Y: 22.6kDa MBP:43.1kDa Reaction is stopped with 5×SDS loading buffer at different time points.
The figure shows the SDS-PAGE analysis of the enzymatic activity of TEV protease on its substrate. The top band corresponds to the intact substrate (MBP-tevS-G-Y), which diminishes over time, indicating substrate cleavage. The lower two bands represent the cleavage products (MBP and G-Y), which increase over time. Samples were taken at 0, 10, 30, 60, 120, and 240 minutes, with the reaction stopped by adding SDS loading buffer and heating at 95°C for 5-10 minutes.
The results confirm that the TEV protease extracted from inclusion bodies is active.
Protein Activity Assay of PPV and split-PPV:
Figure 17: SDS-PAGE analysis of protease activity verification in split and full-length PPV constructs. Figure 17-A：Lane M: Marker; Lane 0: Split-1 at 0 min; Lane Split-1: Split PPVp with 350 nM rapamycin at 10, 30, and 120 min; Lane Split-N: Split PPVp without rapamycin at 10, 30, and 120 min; Lane cPPV: Full-length FKBP-cPPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane nPPV: Full-length FRB-nPPVp with 1400 nM rapamycin at 10, 30, and 120 min. Figure 17-B：Lane M: Marker; Lane Split-2: Split PPVp with 700 nM rapamycin at 10, 30, and 120 min; Lane Split-3: Split PPVp with 1400 nM rapamycin at 10, 30, and 120 min; Lane PPV: Full-length PPVp with its substrate at 10, 30, and 120 min.
The results show that both the full-length PPVp and the split PPVp (when induced by rapamycin) exhibit protease activity, as evidenced by the reduction in substrate over time. In contrast, a single split PPVp cannot cleave the substrate. However, when two split PPVs are combined without rapamycin induction, there is minimal cleavage activity, as indicated by the slight reduction in substrate from 10 to 30 and 120 minutes. The amount of substrate reduction is significantly less than that observed with rapamycin induction, leading to the conclusion that rapamycin is necessary for the optimal activity of the split PPVp.
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We successfully extracted active target proteases from inclusion bodies, but the extraction process is complex and time-consuming. Therefore, we plan to optimize the protocol to enhance the solubility of the target protein and simplify extraction steps.
Cycle 4 - Exploring Tagging Systems
Design
To enhance the solubility of the target proteins, we plan to add solubility tags to the protease sequences. We linked two common solubility tags (MBP and FH8) to each target protein and reconstructed new plasmids with solubility tag-target protein fusions. These plasmids were introduced into BL21(DE3) strains to test whether the solubility of the target proteins had improved.
Figure 18: Effects of Soluble tags. When total proteins are extracted (AU) and centrifuged, the solubility of the proteins can be assessed. Soluble tags are expected to enhance the solubility of the target proteins.
Figure 19: The synthesis pathway and plasmid profiles of MBP-PPVp-pET28a, MBP-FKBP_cPPVp-pET28a, MBP-FRB_nPPVp-pET28a, and MBP-GFP_truncated_linker(PPV)_YFP_truncated-pET28a. The linearized vector was derived from the initial batch of plasmids we constructed for expressing the target protein.
Figure 20: The synthesis pathway and plasmid profiles of FH8-PPVp-pET28a, FH8-FKBP_cPPVp-pET28a, FH8-FRB_nPPVp-pET28a, and FH8-GFP_truncated_linker(PPV)_YFP_truncated-pET28a. The linearized vector was derived from the initial batch of plasmids we constructed for expressing the target protein.
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Using PCR and Gibson assembly, we successfully attached a solubility tag to the initial batch of plasmids we constructed for expressing the target protein, and the sequencing results confirmed its accuracy.
Figure 21: Sequencing results of the MBP-tag and target gene junctions in three plasmids.(A) Sequencing result showing the 6xHis-MBP tag region in all three plasmids. The 6xHis tag is followed by a thrombin cleavage site, and the MBP tag is properly in frame, confirming correct insertion and sequence alignment. (B) Sequencing result of the junction between the MBP tag and the target gene FKBP_cPPVp in MBP-FKBP_cPPVp-pET28a plasmid. (C) Sequencing result at the junction of MBP and FRB_nPPVp in MBP-FRB_nPPVp-pET28 plasmid. (D) Sequencing result of the MBP and GFP_truncated junction in MBP-GFP_truncated_linker(PPV)_YFP_truncated-pET28a plasmid. These sequencing results confirm the correct construction of all three plasmids with the MBP tag and their respective target genes.
Figure 22: Sequencing results of the FH8-tag and target gene junctions in three plasmids. (A) Sequencing result showing the 6xHis-FH8 tag region in all three plasmids. The 6xHis tag is followed by a thrombin cleavage site, and the FH8 tag is properly in frame, confirming correct insertion and sequence alignment. (B) Sequencing result of the junction between the FH8 tag and the target gene FKBP_cPPVp in FH8-FKBP_cPPVp-pET28a plasmid. (C) Sequencing result at the junction of FH8 and FRB_nPPVp in FH8-FRB_nPPVp-pET28a plasmid. (D) Sequencing result of the FH8 and target gene PPVp junction in FH8-PPVp-pET28a plasmid. (E) Sequencing result of the junction between the FH8 tag and GFP_truncated in FH8-GFP_truncated_linker(PPV)_YFP_truncated-pET28a plasmid. These sequencing results confirm the correct construction of all three plasmids with the MBP tag and their respective target genes.
Test
We transformed the newly constructed plasmids into E. coli BL21(DE3) and induced expression of our target proteins. Solubility tests revealed a significant increase in the solubility of all target proteins. Therefore, we proceeded to purify the proteins directly from the supernatant after sonication and centrifugation. Our results showed successful extraction of MBP-cPPV and MBP-G-PPV-Y, while MBP-PPV and MBP-nPPV were not successfully purified due to various factors. However, due to time constraints, we have temporarily paused these experiments and plan to continue them after the Grand Jamboree.
Figure 23: SDS-PAGE analysis of the elution peak of proteins expressed by bacteria through gravity chromatography. Lane1, elution peak of proteins containing MBP-PPV; Lane2, elution peak of proteins containing MBP-nPPV; Lane3, elution peak of proteins containing MBP-cPPV; Lane4, elution peak of proteins containing MBP-G-PPV-Y. The results showed that we had successfully purified MBP-cPPV and MBP-G-PPV-Y, however, we failed to purify MBP-PPV and MBP-nPPV.
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Our results confirmed that adding a solubility tag significantly improved the solubility of the target protein, greatly simplifying the extraction process. We also believe that by increasing the solubility of the target protease, we can directly purify it from the cell lysate supernatant without going through denaturation and renaturation steps, which will likely enhance the protease’s activity. However, due to time constraints, we haven’t yet completed the de-tagging, purification, and activity validation for the tagged protease. We hope to continue these experiments after the Grand Jamboree.
Cycle 5 - Exploring Cell-Free Expression System
Design
To eliminate the impact of purification on protease activity, we used the ALiCE® cell-free protein expression system (CFPS) and redesigned the plasmids for target protein expression. We constructed plasmids for expressing FRB_nPPV, FKBP_cPPV, and PPV proteins in the ALiCE® cell-free protein expression system.
Figure 24: Working principle of the ALiCE® cell-free protein expression system.
Figure 25: Plasmid profiles of pALiCE01_FKBP_cPPVp, pALiCE01_FRB_nPPVp, and pALiCE01_PPVp.
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We used PCR and Gibson assembly to insert the target gene for protein expression into the cell-free expression vector provided by the company, and the sequencing results confirmed its accuracy.
Figure 26: Sequencing verification of plasmids. (A) Sequencing results for the pALiCE01_PPVp plasmid. (B) Sequencing verification of the pALiCE01_FKBP_cPPVp. (C) Sequencing results for the pALiCE01_FRB_nPPVp plasmid.
Test
We used the trial kit from the provider to try to express EYFP protein and performed SDS-PAGE analysis. The result confirmed successful protein extraction with minimal contamination. This demonstrated the potential of the cell-free expression system to produce relatively pure proteins without the need for purification steps.
Figure 27: SDS-PAGE analysis of proteins expressed by the ALiCE ® Cell-Free Protein Expression System. SDS-PAGE analysis of positive control EYFP expressed by cell-free system. Lane2, negative control (ALiCE reaction mixture only) ; Lane3, reaction mixture with EYFP expression; Lane4,reaction mixture diluted two times with EYFP expression; Lane5, reaction mixture diluted twenty times with EYFP expression. We proved that this cell-free protein expression system was feasible.
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The cell-free system showed good performance in the extraction of EYFP protein. However, due to the delay in receiving the full cell-free translation kit from the company, we were unable to validate the expression of our target protease. As a result, this part of the work remained at the plasmid construction and preliminary experiment stages. We plan to complete the remaining experiments after the Grand Jamboree.
Module III: Aptamer-split Protease Crosslinking
Cycle 1 -Click chemistry-based crosslinking
Design
We believe an ideal sensor should be composed of a highly-specialized sensing module, such as an aptamer, and an upstream signal transporter, and in this case, the SPOC system. To conjugate these two mudules, we first employed click chemistry to accomplish crosslinking.
For the aptamers involved in this method, they need to be modified with DBCO. For the click chemistry approach, specifically the p-AzF-DBCO reaction, UAG stop codon site-directed mutations into the cPPVp/nPPVp genes need to be introduced. This modification allows the pEvol-pAzFRS.2.t1 plasmids to incorporate p-AzF through an orthogonal tRNA synthetase/tRNA pair.
Figure 28: Concept diagram of click chemistry-based crosslinking between an aptamer and split protease. The aptamer is modified with dibenzoazacyclooctyne (DBCO), while the split protease contains p-Azido phenylalanine (p-AzF). These components covalently bond when incubated at room temperature, creating a stable complex that recognize biomarkers.
Firstly, plasmids that express the required split proteases were constructed. Specifically, we PCR the nPPVp and cPPVp fragments along with the pET28a backbones from our original plasmids to eliminate the rapamycin sensor components (FRB/FKBP). Then, Gibson assembly was performed to create plasmids containing the split protease genes, which can then be further modified using site-directed mutagenesis via overlap PCR according to the simulation of the dry lab team. Finally, E.coli co-transformed with the mutated plasmids and pEvol-pAzFRS.2.t1 can be induced to express target proteins containing p-AzF.
Figure 29: Overview of plasmid construction for click chemistry-based crosslinking. (A-B) Workflow for the construction of UAG stop codon-mutated plasmids for nPPVp and cPPVp (refer to text for details). (C) Structure prediction of the PPVp protease, highlighting the selected phenylalanine residues for mutation: PHE-6 on nPPVp and PHE-227 on cPPVp. (D) Plasmid mapping of pEvol-pAzFRS.2.t1.
Figure 30: Primers used in click chemistry based crosslinking. Different colors show different uses. Green: primers used to build pET28a_cPPVp plasmid. Blue: primers used to build pET28a_nPPVp plasmid. Yellow: primers used to mutate pET28a_cPPVp plasmid. Grey: primers used to mutate pET28a_nPPVp plasmid.
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Through Gibson assembly, we successfully constructed the pET28a_nPPVp/cPPVp plasmids and the site-directed mutated sequences. The engineering success was proved via sequencing.
Figure 31: Sequencing verification of plasmids. (A-B) Sequencing results for the pET28a_nPPVp plasmid, showing the N-terminal and C-terminal regions, respectively. (C) Sequencing verification of the pET28a_nPPVp_mut, with red boxes highlighting the mutations. (D-E) Sequencing results for the pET28a_cPPVp plasmid, detailing the N-terminal and C-terminal regions, respectively. (F) Sequencing verification of the pET28a_cPPVp_mut, with red boxes indicating the mutations.
Test
We induced protein expression in co-transformed bacteria when the OD600 reached approximately 0.8 by adding IPTG and L-arabinose, along with p-AzF. SDS analysis of total proteins in the cell lysates before ultrasonication was conducted to verify the expression of the target proteins. However, we encountered challenges in purifying the proteins possibly due to low protein yield.
Figure 32: SDS analysis of total proteins in cell lysates before ultrasonication. Lane 2-3: Total proteins from cell lysates of bacteria expressing hCG. Lane 4-5: Total proteins from cell lysates of bacteria expressing nPPV (15.2 kDa) and cPPV (16.8 kDa), respectively.
Thus, we chose to use both AU (after ultrasonication) and SU (supernatant after ultrasonication) components for the protein ligation reaction. Despite employing very gentle warming and cooling during the process, the DBCO-modified aptamer consistently exhibited large molecular weight multimerization bands. Notably, no new bands appeared in the reaction system where the protein was added, indicating that the ligation was not successful.
Figure 33: Protein ligation reaction utilizing click chemistry. 29: DBCO_12T_thrombin_HD22_29mer; 40: thrombin_AYA1809004_40mer_8T_DBCO; 29 Click 1: 29+nPPVp* AU without heating; 29 Click 2: 29+nPPVp* AU with heating; 29 Click 3: 29+nPPVp* SU without heating; 29 Click 4: 29+nPPVp* SU with heating; 40 Click 1: 40+cPPVp* AU without heating; 40 Click 2: 40+cPPVp* AU with heating; 40 Click 3: 40+cPPVp* SU without heating; 40 Click 4: 40+cPPVp* SU with heating.
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During this engineering cycle, we observed that the growth rate of co-transformed bacteria was significantly slower compared to bacteria containing a single plasmid. Given the complexity associated with the incorporation of unnatural amino acids, this may have contributed to a low protein yield, hindering purification efforts. Consequently, no visible shift bands were observed in the protein ligation reaction. As we still got biotin modified aptamer 29/40 from SPR experiments, therefore, we considered utilizing streptavidin-biotin interactions for crosslinking, which will be discussed in the subsequent DBTL cycle.
Cycle 2 - Streptavidin-biotin interactions based crosslinking
Design
Given the challenges associated with the expression of proteins incorporating unnatural amino acids, we are exploring natural bioaffinity systems that can be constructed using simply fused proteins. We have selected streptavidin-biotin interactions for this purpose. Streptavidin (SA) is a tetrameric protein derived from the bacterium Streptomyces Avidini, which exhibits extraordinary affinity for biotin. Streptavidin (SA) can be easily fused with the split protease, and biotinylated aptamers can be readily obtained. This approach simplifies the process while maintaining effective binding and functionality.
Figure 34: Concept diagram of crosslinking between an aptamer and split protease via streptavidin-biotin interactions. The split protease is fused with streptavidin (SA), while the aptamer is biotinylated, allowing for a strong and specific binding that enhances protease functionality.
We firstly constructed SA-fused split protease plasmids. After performing PCR on the target genes and the pET28a plasmid backbone, Gibson assembly will be conducted to generate the desired plasmids. Then we expressed these two proteins in E.coli and used nickel beads to purify.
Figure 35: Overview of plasmid construction for streptavidin-biotin interactions based crosslinking.
However, we were aware of the possible difficulties of extracting the targeted protein considering our experience in the SPOC cycle. Maltose-binding protein (MBP) tag is a large (43 kDa) periplasmic and highly soluble protein of E. coli that acts as a solubility enhancer tag. It also increases protein expression levels[1]. Considering of the large molecular weight of MBP, we were concerned that the MBP tag may interfere with the binding of SA and biotin. So we added a TEV-cutsite between the MBP and SA.
Figure 36: Overview of plasmid construction for streptavidin-biotin interactions based crosslinking.
Build
After the Gibson assembly described above, sequencing was performed to verify the plasmids. The sequencing results confirmed that the construction was successful.
Figure 37: Sequencing verification of plasmids.(A-B) Sequencing results for the pET28a_SA_cPPVp plasmid, showing the N-terminal and C-terminal regions of streptavidin gene, respectively. (C-D) Sequencing results for the pET28a_SA_nPPVp plasmid, showing the N-terminal and C-terminal regions of streptavidin gene, respectively.
Using the same method, we constructed pET28a_MBA_SA_nPPVp and pET28a_MBA_SA_cPPVp successfully. Then we sent the plasmid to sequence and ultimately confirmed that it was correct.
Test
Next, we transferred the correct plasmid into the expression strain of E.coli BL21(DE3). When the culture reached an OD600 of approximately 0.3 at 37°C, IPTG was added, and the induction was carried out overnight at 20°C. However, the SDS-PAGE results showed no significant protein expression.
Figure 38: SDS analysis of the protein expression level and solubility for SA_cPPVp and SA_nPPVp. Lane1-3, total protein, supernatant after sonication, inclusion bodies after washing of SA_cPPVp, respectively. Lane4-6, total protein, supernatant after sonication, inclusion bodies after washing of SA_nPPVp, respectively.
Then, we transferred the correct MBP-fused plasmid into the expression strain of E.coli BL21(DE3). When the culture reached an OD600 of approximately 0.55 at 37°C, IPTG was added, and the induction was carried out overnight at 20°C. Then we did Protein Solubility Verification. We found that our protein was predominantly located in the inclusion bodies, but a portion was also present in the supernatant. The we used nickel beads to purify protein from supernatant. However, there were little target protein in eluate.
So, we can only obtain our protein from the inclusion bodies. Since the majority of the protein in the inclusion bodies is our target protein[2], We directly denatured the washed inclusion body protein using 8M urea, followed by gradient dialysis for refolding. Then we cleaved the refolded protein with TEV overnight.
Figure 39: SDS analysis of the protein purified from supernatant and washed inclusion bodies for SA_cPPVp, SA_nPPVp, MBP_SA_cPPVp and MBP_SA_nPPVp. Lane1-4, protein purified from supernatant. From lane 1 to 4, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively. Lane5-8, washed inclusion bodies. From lane 5 to 8, MBP_SA_cPPVp, MBP_SA_nPPVp, SA_cPPVp and SA_nPPVp, respectively.
We added different amounts of cleaved MBP_SA_nPPVp to 5’biotin-29mer and MBP_SA_cPPVp to 5’biotin-40mer. After incubating at 37°C for half an hour, we performed EMSA to verify the link between protein and nucleic. The EMSA results showed that as the amount of protein increased, the nucleic acid bands darkened. However, no shifted bands were observed.
Figure 40: EMSA analysis of the protein-nucleic linkage. Lane1-4, 5’biotin-29mer and cleaved MBP_SA_cPPVp. Only add aptamer in lane 1. From lane 2 to 5, protein amounts for lane are 0.3ug, 0.6ug, 1.2ug, 2.4ug, respectively. Lane6-10, 5’biotin-40mer and cleaved MBP_SA_nPPVp. Only add aptamer in lane 6. From lane 7 to 10, protein amounts for lane are 0.9ug, 1.8ug, 3.6ug, 7.2ug, respectively. The amounts for aptamer is 20pmol each lane.
Learn
It is challenging to express the split protease with the SA tag alone. Therefore, it is preferable to add a tag that can enhance the expression. Considering the solubility of the protein, we ultimately chose the MBP tag, which had been previously used in the SPOC module.
Our experimental results of MBP-fused proteins preliminarily demonstrated the binding capability between SA-split protease and biotin-aptamer. However, it is strange that no band shift was observed. We suspect this is because the refolded inclusion body protein remains partially aggregated, causing it to get stuck in the wells during gel electrophoresis and not run down. Due to time constraints, we did not conduct further experiments to verify the linkage.
Module IV: Output Module
Cycle 1 - Choosing a Molecule as Output Signal
Design
In the output module, we came up with two methods to convert the split-PPV cleavage event to the final output signal. The methods are all based on the principle of colloidal gold test paper. The colloidal test paper works based on the principle of colloidal particles (typically metals like gold or silver) being immobilized on a test strip to detect specific analytes. The result is often a color change visible to the naked eye. Here’s the method of detecting PPV protease cleavage event.
Firstly, the substrate of PPVp is designed as a fusion protein(GST–PPVp cleavage site–hCG), and the substrate is immobilized on a GST affinity chromatography column. Then, when our sample flows through the column, if the split-PPVp is active in the sample, cleavage happens. hCG flows out into the hCG colloidal test paper. The test paper will show a positive result. If there isn’t any active PPVp in the sample, then the hCG colloidal test paper will show a negative result.
Figure 41: Concept diagram of realizing output with hCG colloidal gold test paper. Fusion protein (GST—PPVp site—hCG) is immobilized on a GST affinity column. Active PPVp in the sample cleaves the substrate, releasing hCG, which is detected by the hCG colloidal test paper, showing a positive result. No cleavage results in a negative test.
Build
Using the method described above, we constructed pET42a_β_hCG_GST successfully. Then we sent the plasmid to sequence and ultimately confirmed that it was correct.
Figure 42: Design of β-hCG-GST plasmid.
Test
The plasmid containing β-hCG-GST was transformed into BL21(DE3) and protein expression was induced. We performed SDS-PAGE analysis of β-hCG, and verified its expression (Fig 3). Then, we tested whether the protein(which was expressed in the supernatant) can trigger the output signal, i.e. the positive band on the colloidal gold test paper. Unfortunatly, there’s no positive band shown.
Figure 43: SDS-PAGE analysis of β-hCG-GST. The black arrow indicates the position of β-hCG-GST.
Learn
Later, we discussed and brainstormed the reason why we failed. What we actually expressed is the β subunit of hCG, but most colloidal gold test kits are for both α and β-hCG, and we can’t find one for β-hCG alone. We plan to further express α-β-hCG-GST fusion protein so that the colloidal gold test paper can produce a positive signal.
R E F E R E N C E
[1] Fox, J. D., Kapust, R. B., & Waugh, D. S. (2001). Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins. Protein science : a publication of the Protein Society, 10(3), 622–630. https://doi.org/10.1110/ps.45201
[2]Singh, A., Upadhyay, V., Upadhyay, A. K., Singh, S. M., & Panda, A. K. (2015). Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial cell factories, 14, 41. https://doi.org/10.1186/s12934-015-0222-8