Engineering

Design2: 'Another biomarker for cervical cancer and its DNA aptamer

'Other Biomarkers':

We explored additional biomarkers that meet the three conditions mentioned earlier: CEA (Carcinoembryonic Antigen) and PTK7.

1. We found the biomarker PTK-7 and its DNA aptamer Sgc-8 [3].

2.We found the biomarker CEA and its DNA aptamers N54, N56, N57, N59, N65, and N71 [4].

[Build2: 'Lateral Flow Assay']

'Chose Different Diagnostic Method'

We decided to use the lateral flow assay (LFA) as the diagnostic method and incorporated it into the sanitary pad.

[Test 2: H-DOCK "Protein-Aptamer Binding Affinity Calculation Program” ]

Among the six DNA aptamer candidates proposed in the reference literature for binding with CEA protein, KUAS-Dry Lab selected the N56 aptamer as the ideal candidate for use in the lateral flow assay (LFA). This decision was based on modeling the CEA N domain & DNA aptamer affinity using the H-DOCK program.

[Result]

Docking score

: more negative docking score means a more possible binding model

Confidence score

: confidence score > 0.7: very likely to bind

0.5 < confidence score < 0.7: the two molecules would be possible to bind

confidence score < 0.5: unlikely to bind)

'N56 aptamr','Sqc-8'&'CEA, PTK7 protein'

Based on the results from the dry lab, we have selected the aptamer N56 with the highest confidence score, which binds to the cervical cancer biomarker CEA. Additionally, we have chosen the aptamer sequence, Sgc-8, which is publicly available in the literature, and its corresponding marker protein, PTK7, to work with.

However, for PTK7, as it is a human protein, its full gene sequence was exceedingly long, exceeding 85,000 base pairs, making it impractical for insertion into a plasmid. We attempted to identify only the domain responsible for interaction with the aptamer, but unfortunately, we couldn't confirm the interaction domain with sgc8. Consequently, we have decided not to use it.

[Experiment DBTL, synthetic biology engineering]

‘CEA expression plasmid design’:

We have constructed a plasmid for the expression of the CEA N domain.

This is because the N56 aptamer binds to the CEA N domain, and the full protein sequence of CEA is too long to be entirely accommodated in a plasmid. Therefore, only the CEA N domain was inserted into the plasmid.

[Design 1: ‘Cell-Free Expression for EMSA’]

We will insert the CEA sequence into a plasmid and use E. coli extract for Cell-Free Expression to induce protein expression. Following protein expression, we will confirm the binding of our selected aptamer to CEA using the Electrophoretic Mobility Shift Assay (EMSA) method. (Design_EMSA)

[Build 1: ‘Cell-free expression’]

We will insert the CEA sequence into a plasmid and use E. coli extract for Cell-Free Expression to induce protein expression. Enzymes present in the E. coli extract induce protein expression from the plasmid even in the absence of cells.

[Test 1 : ’Expression test’ & ‘Purification’ & ‘SDS-PAGE’]

[Learn 1: 'Troubleshooting']

The Cell-Free Protein Expression samples did not indicate significant protein overexpression when comparing between the negative and the samples. This was evident as the expression weight markers where CEA proteins were supposed to be expressed did not show a significant expression signal.

Furthermore, after the purification process, the SDS-PAGE result showed almost no discernible coloration in the bands, suggesting that there was minimal protein presence. Consequently, we concluded that the protein obtained through cell-free expression would not provide a sufficient quantity of protein for conducting EMSA experiments. Therefore, we decided to use cellsfor this purpose.

Additionally, during the purification process, we encountered an issue with the Ni-NTA spin column. It seemed that there was a problem with the column membrane, as during the equilibration step, even after centrifugation, the buffer did not flow down as expected. This issue led to the replacement of the Ni-NTA spin column for negative control samples. As a result, for subsequent steps, we opted to use nickel beads for protein purification to ensure a smooth and efficient process.

[Design 2: 'Protein expression in cells for EMSA.']

We initially attempted protein expression using a cell-free system for CEA, but the protein yield was low, and the target protein was not overexpressed. Due to these issues, we decided to switch to cell-based protein expression to ensure sufficient protein production.

The chosen strain for protein expression: E. coli (DH5alpha_DE3)

1. Versatility: E. coli is a commonly used host organism for protein expression in research and biotechnology due to its well-characterized genetics and the availability of various expression vectors.

2. Rapid Growth: E. coli has a short generation time, allowing for quick production of protein.

3. Strong Promoters: The DH5alpha_DE3 strain contains a T7 RNA polymerase gene under the control of the lacUV5 promoter, which can be induced by adding IPTG (Isopropyl β-D-1-thiogalactopyranoside). This system enables tight control of protein expression.

4. High Yield: E. coli can produce a high yield of recombinant protein when optimized for the specific protein of interest.

[Build 2 : '16°C cell culture’]

1. According to Farewell and Neidhardt (1998, September), as the temperature increases, the elongation rate of protein synthesis consistently shows an upward trend. However, concerning cell growth rate, when it exceeds 37°C, the rate of increase slows down. From this, it can be inferred that excessively high temperatures may lead to the phenomenon where protein synthesis speeds up, but cell growth rate slows down, possibly indicating that ribosomes are unable to synthesize proper proteins. Therefore, when conducting cell cultivation at high temperatures, there is a risk of rapid acceleration in protein expression, potentially resulting in the aggregation of expressed proteins.

2. ‘It is possible that all of these ribosomes are indeed functioning and producing protein but that the rate of protein degradation is greatly increased.’ Farewell, A., & Neidhardt, F. C. (1998, September) Therefore, if the CEA N domain protein is expressed too rapidly, there is a possibility that some of the expressed CEA N domain protein may degrade, resulting in lower protein purity, which may not be suitable for use in EMSA.

For these reasons, we initially cultured the cells at a lower temperature of 16 degrees Celsius to slow down protein expression and prevent aggregation.

[Test 2: 'Expression test']

[Learn 2: ‘CEA protein expression’]

The E. coli culture temperature was too low for efficient CEA protein expression, leading to inadequate expression.

Consequently, we needed to find the optimal temperature for E. coli (DH5alpha_DE3) to express the CEA protein. We conducted E. coli cultures at various temperatures to determine the most suitable one for CEA protein expression.

We also learned that pre-adjusting the temperature before reseeding the cells and then culturing them results in more stable cell growth.

[Design 3: ’Protein expression in cells for EMSA.’]

1. At 16°C, when performing cell culture followed by reseeding and IPTG treatment for expression testing, it was evident that E. coli was too cold for efficient expression of the CEA N domain protein.

2.

In other words, the optimal conditions for cell growth imply having a high concentration of active ribosomes. For this cycle, we set the cell culture temperature at 37°C, which falls within the range of 35-40°C, where E. coli's cell growth rate is the highest. This temperature ensures that our target protein can be produced efficiently in an environment with a high concentration of active ribosomes.

[Test 3-1: ‘Expression test’]

[Build 3-2: ‘30°C cell culture’]

When conducting cell culture at 37°C, there is a risk of rapid acceleration in protein expression, potentially leading to the aggregation of expressed proteins. To mitigate this risk, we also conducted cell culture at a lower temperature of 30°C. 30°C is also a commonly used temperature for cell culture.

[Test 3-2: ‘Expression test’]

[Learn 3-1, 2: ‘Proper Temperature for CEA protein expression’]

Overall, it appears that protein expression was more active at 37°C compared to 30°C. Despite concerns about the high temperature of 37°C potentially leading to protein aggregation during the expression process, we were fortunate not to observe any signs of aggregation on SDS-PAGE. Therefore, we have chosen 37°C as the temperature at which E.coli can express CEA N domain protein vigorously without causing aggregation.

[Design 4: ‘Protein purification for EMSA’]

[Build 4: ‘Protein purification by Nickel bead’]

The initial purification process using Nickel beads

1. After culturing 200 ml of cells, we retrieved the cultured cells and transferred them to four 50 ml conical tubes.

2. In two separate conical tubes, we added 2.5 ml of lysis buffer each and resuspended the cells.

3. After resuspension, we combined the contents of one of the 50ml conical tubes into a single tube, resulting in a total of 5ml of lysis buffer for 100ml of cultured cells.

4. Since our lab didn't have 5ml tubes available, we divided the lysate evenly into 2ml Eppendorf tubes, with each containing 1.6ml of lysate.

5. We added 400 microliters of beads to another 2ml Eppendorf tube and centrifuged it. We discarded the supernatant, which contained the beads, and repeated the wash steps twice using the same buffer used for resuspension.

6. We added 1.6ml of cell lysate supernatant to the washed beads and shook them for 1 hour and 30 minutes.

7. As the lab didn't have a shaking machine, we adjusted an incubation device to 10°C, placed the tubes in ice, and incubated them while shaking.

8. To ensure proper mixing, we manually shook the tubes for 10 minutes.

9. After incubation, we centrifuged the tubes, discarded the supernatant (unbound part sample), added wash buffer, and immediately centrifuged them again. We repeated this wash step three times.

10. Finally, we performed the elution step to release the his-tagged protein (CEA N domain) bound to the beads. We adjusted the imidazole concentration sequentially to 100mM, 250mM, and 500mM to increase elution strength. We extracted the supernatant by centrifuging without any incubation time.

[Test 4: ‘Verifying the purified protein by Coomassie Brilliant Blue G-250 dye’]

[*Coomassie Brilliant Blue G-250 dye*] The Coomassie Brilliant Blue G-250 dye assay is commonly used to detect proteins in a sample. Under acidic conditions, the dye interacts with proteins, causing a shift in color from reddish-brown to blue, which can be measured by absorbance at 595 nm. [8]

When you treated the purified sample with this stain and observed no color change, it indeed suggests that there is either a very low amount or an absence of purified protein in the sample. This conclusion indicates that the purification process did not proceed as expected or may have encountered issues, such as protein loss or insufficient purification.

[Learn 4: ‘Troubleshooting for the purification process, and optimization of protein purification protocol’]

The reasons for the improper purification were as follows:

 1. When preparing the cell lysate, we initially used 5 ml of lysis buffer to resuspend the cells after centrifuging 100 ml of cell culture. However, we divided this lysate into 2ml Eppendorf tubes, resulting in a significant dilution of the expressed protein. Therefore, we concluded that it would be more appropriate to resuspend 100ml of cultured cells with 2ml of lysis buffer in subsequent purification steps.

< 2. During the buffer preparation process, lysozyme and benzonase were accidentally added to both the washing buffer and elution buffer. Originally, lysozyme and benzonase were supposed to act only during the lysis step. Since it was uncertain how lysozyme and benzonase might have affected the purified protein during storage at 4°C after the purification process, it became necessary to remake the buffers.

 3. The step involving the addition of lysis buffer and subsequent centrifugation is the stage where cell lysate is created. In the protocol we referenced originally, it was stated to be done for 20-30 minutes. However, it's important to note that centrifugal force is proportional to the size and rpm (rotations per minute) of the centrifuge. Therefore, even at the same rpm, a larger centrifuge can exert more powerful separation. In our case, we used 2ml E-tubes and a smaller centrifuge, so we adjusted the centrifugation time to 45 minutes for our procedure.

< 4. During the binding process, we inadvertently applied excessive shaking in terms of both duration and intensity. We initially believed that vigorous shaking would lead to better mixing of the beads and protein, resulting in improved binding. Consequently, we applied strong shaking to ensure that the beads remained dispersed and did not settle. However, we soon realized that this excessive shaking could cause even the bound protein to dissociate from the beads. As a result, we decided to borrow a shaking device from a 4°C laboratory to conduct our experiments more effectively.

 5. For each Elution step, it is necessary to have an incubation period of about 10 minutes before centrifugation. Although it was not mentioned in the protocol we initially referred to, we decided to implement this step to improve protein purification. We believed that allowing sufficient time for imidazole in the elution buffer to bind to the beads and facilitate protein detachment was crucial for successful purification.

[Design 5: ‘Purifying the protein anew for EMSA’]

[Build 5: ‘Protein purification by Nickel bead’]

[optimized protein purification protocol]

We conducted protein purification using a protocol that had been optimized to suit our experimental needs.

For laboratories lacking column tubes specialized for protein purification, centrifuges compatible with 5ml tubes, or specialized protein purification kits, our protocol can serve as a valuable reference if they have access to a 2ml tube-compatible centrifuge and nickel or cobalt beads for purifying his-tagged proteins.

Our method involves centrifugation followed by pipetting the supernatant, making protein purification feasible even in resource-constrained laboratory settings.

We believe this information can be beneficial for individuals facing similar equipment and resource limitations.

For more detailed information, you can refer to the experiments section on the KUAS_seoul wiki.

[Test 5: ‘Verifying the purified protein by Nanodrop’]

Most of the proteins that do not bind to the beads are extracted in the unbound step, resulting in the highest protein concentration. As we progress through the washing steps, such as wash 1, wash 2, and wash 3, proteins weakly bound to the beads should be washed away, leading to lower protein concentrations.

In the elution steps, elution 100 (imidazole concentration: 100mM), elution 250 (imidazole concentration: 250mM), and elution 500 (imidazole concentration: 500mM), protein concentrations should increase progressively.

In general, since the description aligns with the expected pattern, it can be inferred that purification has been to some extent successful. However, for a more precise assessment of the purification outcome, it is necessary to prepare samples with a consistent amount of purified protein and run SDS-PAGE gel electrophoresis.

[Learn 5:'Troubleshooting']

After conducting the Nanodrop analysis, we determined the protein concentrations in the samples. Utilizing this information, we standardized the concentrations of the samples to ensure uniformity for an accurately controlled protein purification process.

However, upon further reflection regarding the overall low concentration, we realized that this was likely because we initially used only 100ml of cell culture when preparing the 2ml lysis buffer and conducting the purification. Consequently, to increase the protein concentration, we learned that it would be more effective to use 200ml of cell culture in the 2ml lysis buffer.

Additionally, the SDS-PAGE result shows that contamination was responsible for the observed outcomes. We opened the possibility that an E-tube from outside the lab, rather than sterilized E-tubes designated for the clean bench, was accidentally used to store the protein purification sample. This may have led to contamination in the sample, and it is possible that contamination occurred during the SDS sample preparation process as well.

[Design 6: ‘Purifying the protein anew for EMSA’]

[Build 6: ‘Protein purification by Cobalt bead’]

Generally, cobalt beads are noted to have higher specificity and lower affinity for His-tagged proteins compared to nickel beads. The active center of a nickel-based resin exhibits a more lenient spatial preference, allowing it to interact with histidine residues located in positions other than the protein's polyhistidine tag. This results in increased yields but lower levels of purity.

Hence, we aimed to improve the purity of the CEA N domain protein by changing the type of beads used for protein purification. We maintained the same buffer composition and protocol throughout the process.

[Test 6: ‘Verifying the purification of protein by Nanodrop’]

Most of the proteins that do not bind to the beads are extracted in the unbound step, resulting in the highest protein concentration. As we progress through the washing steps, such as wash 1, wash 2, and wash 3, proteins weakly bound to the beads should be washed away, leading to lower protein concentrations.

In the elution steps, elution 100 (imidazole concentration: 100mM), elution 250 (imidazole concentration: 250mM), and elution 500 (imidazole concentration: 500mM) were applied, in which the protein concentrations should also increase progressively.

In general, since the description aligns with the expected pattern, it can be inferred that purification has been to some extent successful. However, for a more precise assessment of the purification outcome, it is necessary to prepare samples with a consistent amount of purified protein and run SDS-PAGE gel electrophoresis. This part of the experiment is still ongoing.

[Learn 6: ‘CEA protein expression and purification optimization.’]

Through the DBTL process aimed at achieving the optimal expression of the CEA N domain protein, we have come to several key realizations:

 1. Choosing a Different Strain One crucial insight is the importance of going back to the beginning and selecting a different bacterial strain. We believe that transforming the desired plasmid into E. coli BL21, a strain specialized for protein expression, could yield larger quantities of the protein. In contrast to E. coli DH5α DE3, which exhibits lower protein expression levels, using nickel beads for purification instead of cobalt beads is more appropriate, as it allows for higher protein yields.

 2. Optimizing Culture Temperature: When using the BL21 strain, fine-tuning the culture temperature may be necessary to achieve the best results. Temperature conditions around 30-37°C seem to be suitable, but further optimization is likely needed.

 Utilizing the Entire Culture To increase the concentration of purified protein, it's essential to utilize the entire 200ml cell culture during the protein purification process.

In the next cycle, we plan to build upon these lessons learned and conduct a completely new protein purification process. We will then proceed with EMSA experiments to determine whether the purified protein effectively binds to the aptamer, leveraging the knowledge gained during the learning process.

[Design 7: ‘EMSA: test of the binding of protein and aptamer.’]