Part:BBa_K4953000

CEA N domain

This part is based on the CEA N domain sequence information revealed by Abdul-Wahid et al.[1]. The CEA protein is a biomarker for certain cancers, including colon and cervical cancer, and it is detectable in the serum [2,3]. This protein holds great promise as a biomarker in cancer research.

We conducted both Cell-free expression (CFE) and cell expression experiments to produce the CEA protein using this part. While the CFE experiments were not successful, we confirmed the expression of the protein in cell expression. Optimization of expression conditions and other factors may be necessary to utilize CFE effectively.

Although further investigation is needed for successful expression under cell-free conditions, we have verified that the protein is expressed sufficiently within cells.

This part can be valuable for future iGEM teams interested in cancer diagnosis, treatment, and related fields. It allows for the easy expression of the CEA protein when needed and opens the possibility of expressing CEA proteins with specific accessory proteins for their projects.

[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.

<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 cells for 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.’>

<Build 3-1: ‘37°C cell culture’>

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.

‘The optimal temperature for E. coli growth is usually considered to be 35–40°C.’ Chih-Yu Yang et al. (2020) ‘The growth rate depends on the rate at which the cell is able to synthesize new proteins 6,12. This, in turn, depends on the cellular ribosome concentration, but also on how efficient each ribosome is used.’ (Bosdriesz et al., 2015)

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.

[Reference]

[1] A. Abdul-Wahid et al., “Induction of antigen-specific TH9 immunity accompanied by mast cell activation blocks tumor cell engraftment,” International Journal of Cancer, vol. 139, no. 4, pp. 841–853, Apr. 2016, doi: https://doi.org/10.1002/ijc.30121.

[2] S. Dasari, R. Wudayagiri, and L. Valluru, “Cervical cancer: Biomarkers for diagnosis and treatment,” Clinica Chimica Acta, vol. 445, no. 7–11, pp. 7–11, May 2015, doi: https://doi.org/10.1016/j.cca.2015.03.005.

[3] V. L. Kankanala and S. K. R. Mukkamalla, “Carcinoembryonic Antigen,” PubMed, 2022. https://www.ncbi.nlm.nih.gov/books/NBK578172/

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 320
  Illegal XhoI site found at 441
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]