1. Plasmid Design
For our plasmid design, we chose pET-21a(+) due to its strong expression ability in E. coli. It consisted of:
Key Plasmid Components
- Ampicillin Resistance (ampR): This allowed us to select for transformed E. coli cells by growing them in ampicillin.
- LacI Repressor: We included LacI to control gene expression. LacI binds to the lac operator, preventing transcription until we induce it with IPTG.
- Lac Operator: The lac operator ensures tight regulation by blocking transcription in the absence of IPTG. IPTG deactivates LacI, allowing expression to occur.
- Ribosome Binding Site (RBS): The RBS ensures efficient translation of the gene, increasing protein yield after induction.
- LacI Promoter: The lacI promoter drives the expression of the LacI repressor, maintaining control over when the gene is expressed.
- Origin of Replication (ori): The pBR322 ori allows high-copy replication of the plasmid in E. coli, ensuring enough plasmid and protein are produced.
- Balcp19k gene: We inserted our target gene in the MCS.
Overall, this design gives us precise control over protein expression, maximizing yield and minimizing cell stress.
Sequence Optimization
For the cp19k coding sequence, we originally found a biobrick on the iGEM registry. However, we noticed that its CG content was not within the optimal range of 40-60% for best expression in E. coli. To combat this, we researched literature and found an optimized sequence in Xu ZhenZhen, 2020, which optimized CG content to 50%, the resulting gene a total of 522bp. Then we added a 6xHis tag and a start and stop codon to make a coding sequence of 549 bp, which we named Balcp19k and submitted to the registry as part BBa_25FRPPH9. This was sent to the company Nanjing Genscript where the plasmid was sequenced and mailed back to our school.
2. Transformation of Plasmid
Following the protocol from the iGEM website with modifications (see Experiments, Part 1), we transformed the sequenced plasmids to BL21 (DE3) competent cells.
Then, we spread the transformed bacteria on agar plates with AmpR.
The transformation of the plasmid was successful and we had bacterial growth on agar plates with Ampicillin resistance (top plate) and control (bottom right). For the inoculation, we extracted the individual colonies marked with green on the top plate.
3. Inoculation
We inoculated (following the protocol in Experiments, Part 2) the transformed cells in LB medium and left them overnight in a shaking incubator at 37 degrees celsius, 250rpm. The bacteria had successfully grown in 4 of the 5 tubes (tube 1, 3, 4, 5 in the image above), as evidenced by the turbidity/cloudiness of the solution.
4. Glycerol Stock Storage
Following our successful inoculation, due to our limited time frame, we had to halt the experiments over the summer and prepare the glycerol stocks for long term storage. We combined 600 microliters of the overnight bacterial culture with 400 microliters of 50% glycerol solution in cryotubes. These stocks were stored at –80 degrees celsius, allowing us to preserve our stock over the summer holiday.
5. Re-growth of Bacteria (After the Summer)
After the summer break, we revived our transformed cells and retrieved the glycerol stocks from the freezer where we thawed them. A small sample was used to inoculate fresh LB medium containing ampicillin which was then stored overnight in a shaking incubator at 37 degrees celsius. This successfully regrew the bacteria and provided the needed active bacterial culture needed for the next steps of protein expression.
6. Induced Expression with IPTG
After confirming successful transformation and regrowth of our engineered E. coli BL21 (DE3) cells containing the pET-21a (+)-Balcp19k plasmid, we proceeded to induce protein expression of the cp19k gene using IPTG as the inducer under the T7 promoter system.
Procedure
We first diluted overnight cultures 1:100 into 5 mL of fresh LB medium containing ampicillin (100 µg/mL) to maintain plasmid selection. The cultures were incubated at 37 °C with shaking at 220 rpm until the optical density at 600 nm (OD₆₀₀) reached 0.6, corresponding to the mid-logarithmic growth phase of the optimal point for protein induction. We used a spectrophotometer to measure OD₆₀₀ value of 0.61, indicating healthy bacterial growth and a suitable cell density for induction.
At this point, IPTG was added to a final concentration of 1.0 mM to initiate transcription from the T7 promoter by inactivating the LacI repressor. The culture was then transferred to 27 °C and incubated for 4 hours with shaking at 220 rpm to promote soluble protein expression and reduce inclusion body formation, which can occur at higher temperatures.
After induction, 1.5 mL of the culture was harvested by centrifugation at 10,000 rpm for 5 minutes. The supernatant was safely discarded, and the resulting cell pellet was resuspended in 400 µL PBS for downstream protein analysis.
Key Results
This step successfully marked the induced expression phase of our cp19k protein. The recorded OD and pellet formation confirmed healthy post-induction growth, suggesting that protein expression occurred as expected. The samples were subsequently prepared for Ni-NTA purification and SDS-PAGE analysis, described in the following section.
7. Purification with Ni-NTA Column
After successfully inducing expression, protein should be in the cell pellet. The cell pellet is then resuspended in Lysis buffer. This is incubated on ice for 30 minutes and centrifuged at 14,000x for 30 minutes at 4 degrees. The resulting supernatant will contain the soluble cement protein. A 5µL aliquot was taken and added with 5µL of SDS page buffer, to run a trial of SDS page, confirming whether the protein was present. In the first trial, there were no visible bands, presenting a lack of protein. We hypothesized the reason being that a low volume of E. coli cell pellet was used so another 250ml inoculation was started in an attempt to obtain more protein.
Second Trial - Improved Protocol
The 250ml inoculation was centrifuged to extract the E. coli pellets and resuspended in lysis buffer for the second trial of protein purification. The supernatant containing the protein is filtered through a Fast start column and the flow through is collected as the first fraction. The column is further washed twice with 4ml Wash buffer and the flow through was collected again for the 2nd and 3rd fraction. The column is washed twice more with 1ml Native elution buffer to form the 4th and 5th fraction.
Bradford Assay Results
The concentration of protein of each fraction is analyzed using the Bradford Assay, where all fractions yielded proteins, presenting success. A calibration curve was created with increasing concentration of protein, so the absorbance of each fractions can be used calculate the concentration of protein in them. The fraction with the most protein was the 2nd elution wash (5th fraction) with 0.2mg/L. So this fraction was decided to be used for the enzyme protein digest, as it contained the most protein.
7. SDS-PAGE Analysis
The amount of protein in each fraction is analyzed further by running an SDS page.
The second trial showed visible bands which is promising, showing that protein was present. It confirms the error in the first trial where total E. coli pellet used was too small to produce enough protein. The SDS page was "smiling" so we had adjusted our technique using a more appropriate voltage in following gels and the issue was resolved.
8. Protease Digest
After the purification of CP-19k using the Ni-NTA, individual samples were incubated with proteases alcalase and trypsin at 37ºC overnight to test their respective efficacies.
Analysis of Purification Results
The final fraction of the Ni-NTA (elution 2) yielded a moderately impure sample, with 10 bands at different molecular weights. This suggests the solution was contaminated with co-eluted proteins, indicating insufficient washes during the purification process (GoldBio). The most prominent bands had molecular weights of 35.4, 26.9 and 13.4 kDa, rather than the anticipated 19kDa. This may have been because SDS pages utilise total protein charge as a migration mechanism, meaning that it is not highly sensitive to molecular weight, but rather the charge of SDS-bound residues. Disordered proteins, or proteins that have undergone glycosylation, methylation can all alter the presented molecular weight. (Nature) (PMC)
However, this discrepancy could also be caused by the presence of formed disulphide bonds which may have been prevented by the addition of ß-mercaptoethanol. (Novagen)
Alcalase Results
The two alcalase samples varying results. The first alcalase sample had a band at an initial 36.2 kDa as well at lower molecular weights of 17.2 and 13.1 kDa. This meant that the alcalase had cleaved the original protein into significantly smaller fragments. There appear to be no bands above 36.2 kDa meaning that the protein concentration of the sample was either too small or the fragment concentrations were too low to be visible. The second sample had yielded a blank lane, which could mean that the final aliquot collected had a loading sample with too low of a protein concentration.
Trypsin Results
Trypsin was able to digest the prominent 35 kDa band and higher molecular weight bands, creating a secondary band at lower molecular weights at 31.6 kDa in both samples. This suggests that the enzyme was able to catabolise the protein, but not fully as there were still bands at 34.7 and 35.6 kDa. This proves that the protease has disputable efficacy but is unlikely to have targeted the desired protein.
Discussion and Future Directions
Analysis of Ambiguities
These ambiguities suggest there were human errors (e.g. problems with loading the wells), unsuitable method choice or, measurement error, and an inadequate sample size. The methodology also could not provide the definite uncertainty of each band's molecular weight as it is relative to the protein ladder standard and is reliant on the clarity of the original .TIF file.
Recommendations for Future Work
An ideal cp-19k-ase would have a high affinity and specificity and could potentially be modelled off of certain areas of alcalase's active site. This confirms the hypothesised efficacy of proteases and cleavage sites in silico. If these experiments were to be repeated, a purer starting sample should be used, and another method of quantifying protease catabolism efficacy should be considered (e.g. thin layer chromatography, mass spectrometry and UV/Vis spectroscopy) (ScienceDirect) (ACS Omega)