Colony PCR
Design
As part of our wet lab process to confirm successful cloning of our constructs and transformation into bacterial cells, we needed to perform colony PCR. In theory, this is a straightforward process: a PCR reaction mix is prepared according to protocol, but instead of template DNA, a tiny piece of a transformant colony—which should contain the successfully cloned gene fragment—is used. After PCR and gel electrophoresis, we expect to see a band of amplified DNA at the correct length.
Build
After cloning and transforming our plasmids into bacterial cells, the cells were incubated for up to 48 hours. Five colonies from each transformant were randomly selected to test for successful cloning. Colony PCR was performed according to the protocol, and the products were run on an agarose gel.
Test
The electrophoresis gel was visualised and analysed.
Learn
On the first colony PCR gel, we observed a large, glowing cloud in the region where we expected the amplicon DNA band. Even if the amplicon band was present, the glowing cloud obscured it. We hypothesised that this cloud might be cellular debris or other contaminants left over from cell lysis during the PCR. To address this, we theorised that centrifuging the PCR tubes could pellet any insoluble debris.
Figure 1. First colony PCR, a large, glowing cloud can be observed for each well. Picture is cropped to better visualise the problem.
Design
One challenge was that we lacked a centrifuge capable of spinning 0.2 mL PCR tubes at the required speed. To solve this, we designed 3D-printed adapters that would fit into a standard centrifuge carrier for 2 mL tubes.
Build
The adapters were 3D-printed. Colony PCR was repeated, and this time, before gel electrophoresis, the tubes were centrifuged to sediment any debris. The supernatant was then loaded onto the agarose gel, and electrophoresis was performed.
Test
The electrophoresis gel was visualised and analysed.
Learn
Unfortunately, the glowing cloud persisted, indicating that centrifugation did not resolve the issue. After consulting our supervisor, it was suggested to add RNase to the PCR mix to degrade any RNA present.
Figure 2. Expression at 37 degrees 4 h VS 20 degrees overnight. BCBI - BL21(DE3) cells before induction; WCL - whole cell lysate; LS - lysate supernatant; CD - cell debris.
Design
A revised colony PCR protocol was designed, incorporating additional magnesium dichloride and RNase.
Build
The new protocol was tested on the same colonies, but on a smaller scale, to evaluate the effect of adding RNase. PCR was performed, and samples were loaded onto the gel—this time without centrifugation.
Test
The electrophoresis gel was visualised and analysed.
Learn
Analysis revealed that the "cloud of doom" was indeed RNA, as the addition of RNase eliminated the cloud. The gel was clear, and the DNA amplicon bands were clearly visible. Although, further on different primers were also used, which produced a longer amplicon, and wouldnt have been in the RNA cloud anyways.
Figure 3. Colony PCR when RNase was added as a part of the PCR mixture. Lack of a glowing cloud can be observed, and clear amplicons are visible.
Hydrogels
Design
For hydrogel preparation, we decided to follow a protocol described in F.song et al., 2009 [1]. This protocol specified a final casein concentration of 8% w/w, with three genipin concentrations: 2.5 mM, 5 mM, and 10 mM. It also recommended crosslinking at 50°C to accelerate the process.
Build
We followed the protocol, but many details were ambiguous (e.g., where to dissolve genipin), and several steps required assumptions. Despite this, we prepared the hydrogel mixture, pipetted it into HPLC vials, and incubated it at 50°C for the suggested duration.
Test
After incubation, we inverted the vials to check for hydrogel formation. Unfortunately, the mixture remained as runny as before, with no increase in viscosity—though it had turned dark blue.
Learn
No hydrogel formed, so no further tests could be performed. However, the color change from transparent to dark blue suggested a reaction between genipin and casein proteins had occurred. The exact reason for the lack of gelation remained unclear.
Figure 4. Hydrogel mixture before incubation.
Figure 5. Hydrogel mixture after incubation. Solutions were still
liquid, but a clear colour change was observed.
liquid, but a clear colour change was observed.
Design
We hypothesized that the crosslinking time might have been insufficient, so we decided to repeat the experiment with an extended incubation period of 24 hours.
Build
The hydrogel mixture was prepared as before, with the only change being the increased incubation time.
Test
After 24 hours, we again inverted the vials to test for hydrogel formation. The mixture was still liquid, though the same dark blue color change was observed.
Learn
The test for hydrogel formation failed once more. The mixture remained non-viscous, and no conclusions could be drawn beyond the color change. We suspected that the protocol’s lack of specific details might have contributed to the failure.
Design
Next, we switched to a different protocol N.F.N. Silva et al., 2014 [2]. This time, we dissolved a lower concentration of casein in HEPES buffer with added calcium chloride, adjusted the pH to 7.10, and prepared genipin in HEPES buffer with absolute ethanol to a concentration of 200 mM. The final concentrations in the hydrogel mixture were set to 25 g/L casein and 5 mM, 10 mM, or 20 mM genipin.
Build
We followed the new protocol precisely, without needing to guess any steps or values. The hydrogel mixture was prepared in HPLC vials and incubated in a 50°C water bath for 24 hours.
Test
After incubation, we performed the vial inversion test again. Disappointingly, the mixture remained liquid, despite the same transparent-to-dark-blue color change.
Learn
From these tests, we concluded that forming hydrogels from casein and genipin is more complex than initially anticipated. After further literature review, we suspect that sodium caseinate may not be suitable for hydrogel formation using genipin. Instead, assembled casein micelles or differently processed casein proteins might be required.
Figure 6A. Hydrogel mixture before incubation.
Figure 6B. Hydrogel mixture after incubation.
Solutions were still liquid, but a clear colour change was observed.
Solutions were still liquid, but a clear colour change was observed.
Additional Test for Crosslinking
As an additional test, we performed SDS-PAGE under non-reducing conditions to assess potential crosslinking. If crosslinking had occurred, we expected to see an increase in molecular weight. However, the results were inconclusive: while some staining was observed, it remained at the start of the separating gel. This could indicate extensive crosslinking, preventing the proteins from entering the gel—but such a conclusion is unreliable.
Figure 7. SDS-PAGE performed under non-reducing conditions.
Next Steps
Further research is needed, including more extensive literature review, potential re-micellisation of casein, or alternative procedures.
Propolis antimicrobial property tests on E. coli and S. cerevisiae
Design
For antimicrobial testing, propolis was extracted using PEG 400 to avoid ethanol-related growth inhibition. The concentration of phenolic compounds was assumed to be 10.7 ± 1.2 mg/mL, based on a publication from which the protocol for propolis extract was sourced L. Kubiliene et al., 2015 [3]. Dual-layer agar plates with agar well diffusion methods were employed, following the same publication.
Build
We prepared the propolis extract and inoculated plates with E. coli and S. cerevisiae (separately). Four wells were punched into the agar layer, and 100 µL, 150 µL, and 200 µL of propolis extract were pipetted into the wells on separate plates. The plates were incubated for 48 hours.
Test
After 48 hours of incubation, the plates were scanned and analysed digitally.
Learn
In the scanned images, no inhibitory effect from propolis was observed. While observing the plates with the naked eye, we noticed a slight reduction in microbial growth, but not enough for any meaningful analysis. We concluded that this method might not be suitable for our needs, especially since we were unable to precisely determine the phenolic content of our propolis extract due to a lack of proper tools. Additionally, we theorised that liquid extract diffusion from agar wells might be too slow because the plates were freshly poured.
Figure 8. Scan of dual-layer agar plates with 4 wells for propolis diffusion. Top right and bottom right: plates inoculated with E.coli. Top left and bottom left: plates inoculated with S.cerevisiae.
Design
Based on the previous iteration, we decided to repeat the experiment using plates that had been left to dry slightly in an aseptic environment, allowing the liquid propolis extract to diffuse better and faster. A positive control with sterile 20% PEG 400 solution—the same solution used for preparing the propolis extract—was also added.
Build
The same protocols were followed, incorporating the changes mentioned above.
Test
After incubation, the plates were scanned for digital analysis.
Learn
Unfortunately, the results did not differ from the previous experiment. No visible inhibition of microbial growth was observed around the agar wells containing propolis extract or the 20% PEG 400 solution. As before, slight growth reduction was noticed with the naked eye, but it was insufficient for analysis. We decided to try another inoculation method using a colony assay stamp we had designed and 3D-printed. Instead of the dual-layer agar method, we would use agar plates with colonies stamped in a specific arrangement, featuring a central well for the propolis extract.
Design
As mentioned, we used the 3D-printed colony assay stamps to assess the antimicrobial effect of propolis. Other methods remained unchanged.
Build
The colony assay stamps were prepared for use. Plates that had been left to dry in an aseptic environment were inoculated with the respective microorganism using the stamps. A central well was punched out and filled with propolis extract. The plates were incubated for 48 hours.
Test
After 48 hours of incubation, the plates were observed and scanned for documentation.
Learn
This time, smaller colonies were observed closer to the well containing propolis extract, with larger colonies appearing farther away, especially at the edges of the colony assay. While this suggested a potential antimicrobial effect, we also considered that nutrient competition among colonies might have contributed to the reduced growth of inner colonies. For further testing, we decided to use 96-well plate assays to improve repeatability and data reliability.
Figure 9. Colony assay with a central well for propolis diffusion.
Test on S.cerevisiae.
Test on S.cerevisiae.
Figure 10. Colony assay with a central well for propolis diffusion.
Test on E.coli.
Test on E.coli.
Design
The 96-well plate assay was designed to test the antimicrobial effect of propolis at different concentrations. This approach would allow us to quantify the results and determine the amount of extract needed for an antimicrobial effect in liquid culture. To account for any inhibitory effects from the 20% PEG 400 solution, control repetitions were included using the PEG solution at the same volumes as the propolis extract. A negative control with only media (and the same volume of extract solvent) was also added.
Build
The 96-well plates were prepared and filled with the respective propolis extract concentrations. After inoculation, the plates were placed in a plate reader.
Test
Optical density data over time was measured and analysed using the plate reader.
Learn
Analysis revealed that higher concentrations of propolis extract correlated with increased inhibition of bacterial growth, confirming the presence of antimicrobial compounds in our extract. However, the inhibitory effect was weaker than expected. Increasing the extract volume was considered, but its opacity would interfere with optical density measurements, compromising data reliability. To address this, we proposed producing stronger extracts by using more propolis.
Figure 11. 96-well plate assay for testing propolis antimicrobial properties. S.cerevisiae in the top plate, E.coli in the bottom plate. Assays pictured after the experiment.
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Figure 12. Growth curves of S.cerevisiae in presence of increasing concentrations of Propolis and PEG solution.
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Figure 13. Growth curves of E.coli in presence of increasing concentrations of Propolis and PEG solution.
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Protein expression
Design
To produce our proteins of interest, an E. coli expression system was selected due to its typically high protein yield, short expression time, and low production cost. Moreover, protein production in E. coli is easily scalable, allowing to express the proteins in analytical amounts for the evaluation of various expression conditions to identify the optimal parameters and later scale up to obtain higher amounts of protein of interest for further applications. For protein production the canonical BL21(DE3) was chosen as a reliable option for recombinant protein expression.
Build
BL21(DE3) competent cells were transformed with plasmid constructs containing genes coding for proteins of interest. For protein expression, transformed cells were cultivated at 37°C, shaking at 180 rpm, induced with 1 mM final concentration of IPTG at OD600 0.6 - 0.9 and cultivated in the same conditions for 4 h afterwards.
Test
Small-scale protein expression and solubility tests were performed to evaluate protein expression efficacy in described conditions. Results were analysed by SDS-PAGE. We managed to successfully express the recombinant α-S1-casein containing 6xHisTag and TEV-site.
Learn
We learnt that our α-S1-casein is being expressed by the engineered E. coli, however, it is highly insoluble. At this point we started to look for optimisation possibilities for protein expression protocols to enhance soluble protein expression.
Figure 14. Analysis of α-S1-casein production efficiency and solubility by SDS-PAGE. Protein was expressed in E. coli BL21(DE3) at 37 °C for 4 hours.
Design
During research we found out that lowering the expression temperature and final IPTG concentration for induction promotes slower protein expression, which often results in higher yields and improved solubility of the proteins produced. For this reason it was decided to apply a protein production optimisation protocol for α-S1-casein expression in hopes of improving soluble protein expression and possibly obtaining higher protein yields
Build
BL21(DE3) cells that were previously transformed with our plasmid constructs were cultivated at 37 °C, shaking at 180 rpm until the OD600 reached 0.4 - 0.5, after which the temperature was lowered to 20°C, the cell culture was cooled, and protein expression was induced with 2x lower final concentration of IPTG at OD600 0.6 - 0.9. Protein production was carried out overnight.
Test
Protein production efficiency and solubility were tested once again to evaluate the effect of protocol modifications. The results were analyzed by SDS-PAGE.
Learn
We learnt that lowering the expression temperature indeed optimises protein production and yields a higher overall protein content. However, it did not contribute much to solubilising the recombinant α-S1-casein.
Figure 15. Analysis of α-S1-casein production efficiency and solubility by SDS-PAGE. Protein was expressed in E. coli BL21(DE3) at 20 °C overnight.
Design
Since the bovine α-S1-casein produced was mostly insoluble with a small soluble protein fraction in relation to the overall amount of protein produced, we decided to use a different strain of E. coli to see if we can manage to increase the fraction of soluble proteins produced by the E. coli. For that we used an E. coli strain, Rosetta 2, which is a derivative of BL21 (DE3) optimised for expression of eukaryotic proteins.
Build
A new successful transfection was performed, using the same α-S1-casein gene-containing plasmid construct and competent Rosetta 2 E. coli cells. Since earlier experiments clearly showed the advantage of expressing proteins in E. coli at lower temperature overnight, protein production experiments in the Rosetta 2 strain were carried out in the same conditions – with induction of 0.5 mM final concentration of IPTG and protein expression overnight at 20°C with shaking at 180 rpm.
Test
Protein production efficiency and solubility after expression in the E. coli Rosetta 2 strain were tested to evaluate if using an alternative strain improves the solubility of the protein and offers comparatively better yields of soluble proteins. Analysis was done by SDS-PAGE.
Learn
We found that using Rosetta 2 cells for the expression of α-S1-casein offers a similar overall protein yield compared to the BL21(DE3) system in the same expression conditions, indicating that this strain is also suitable for producing our protein of interest. However, it did not significantly contribute to solubilising the α-S1-casein, as no notable improvement was observed in the amount of soluble protein obtained. Protein expression is happening at a high level, although additional optimisation steps are required to increase the solubility. One of the possible approaches would be to introduce solubility tags and test if that has any positive impact on the outcome of protein expression
Figure 16. Comparative analysis of α-S1-casein production efficiency
and solubility by SDS-PAGE in BL21 (DE3) strain at 37 °C, in BL21(DE3) strain at 20 °C and Rosetta 2 strain at 20 °C. Protein expression at 20 °C significantly improves the production yield, however no notable improvements are detectable for the increase of soluble protein fraction.
and solubility by SDS-PAGE in BL21 (DE3) strain at 37 °C, in BL21(DE3) strain at 20 °C and Rosetta 2 strain at 20 °C. Protein expression at 20 °C significantly improves the production yield, however no notable improvements are detectable for the increase of soluble protein fraction.
References
[1] F. Song, L.-M. Zhang, C. Yang, and L. Yan, “Genipin-crosslinked casein hydrogels for controlled drug delivery,” International Journal of Pharmaceutics, vol. 373, no. 1–2, pp. 41–47, Feb. 2009, doi: 10.1016/j.ijpharm.2009.02.005.
[2] N. F. N. Silva, A. Saint-Jalmes, A. F. De Carvalho, and F. Gaucheron, “Development of Casein Microgels from Cross-Linking of Casein Micelles by Genipin,” Langmuir, vol. 30, no. 34, pp. 10167–10175, Aug. 2014, doi: 10.1021/la502274b.
[3] L. Kubiliene et al., “Alternative preparation of propolis extracts: comparison of their composition and biological activities,” BMC Complementary and Alternative Medicine, vol. 15, no. 1, May 2015, doi: 10.1186/s12906-015-0677-5.