Part:BBa_M36763:Experience

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Applications of BBa_M36763

Performance Data:

Transforming plasmid into S. cerevisiae

The chicken lysozyme in yeast chimera plasmid was transformed into yeast cells via PEG-mediated yeast transformation. After plating the yeast onto synthetic complete, leucine drop out plates, the samples were incubated at 30 degrees Celsius for three days. There was high transformation efficiency with an average of 30 yeast colonies per plate. Three colonies were selected for overnight culture in synthetic complete with leucine drop out media. 20% glucose was added to some cultures to repress the promoter and 20% galactose was added to some cultures to induce the promoter. Sucrose was used as the neutral carbon source.

Agar Diffusion Assay for Initial Lysozyme Screen

Agar diffusion plates were set up with with Bacto agar and 30 g/L Micrococcus lysodeikticus (also known as Micrococcus luteus) suspended in 67 mM phosphate buffer. Using the top of P200 pipet tips , holes were punched into the agar and the three cultures (induced, repressed, and nothing) were added the wells. Untransformed, wild-type yeast were used as the negative control and 0.1 g/L chicken lysozyme from Sigma-Aldrich was used as the positive control. The plates were incubated at room temperature for 24 hours. Ideally, if lysozyme is being made and secreted, it will diffuse out of the well and lyse the Micrococcus, thereby clearing a halo around the well. None of the experimental wells showed any clearing. The positive control showed significant clearing - about 3 mm from the well - while the negative showed none, as expected. We reasoned that the lysozyme must either be produced at very low concentrations, have a low Vmax, or is not being secreted properly. In the next diffusion assay, the cultures were lysed and the supernatant was added to the wells. Again, there was no clearing around the wells. This led us to suspect that the lysozyme is not very active and/or it is being produced at low concentrations.

Kinetic OD Assay

To quantitatively measure the activity of lysozyme, a plate assay was conducted where 100 ul of 1 g/L M. lysodeikticus was added to each well with 10 ul of enzyme. The plate was read over a period of 45 minutes at 450 nm. The negative control was substrate and buffer while the positive control was substrate and 0.1 g/L chicken lysozyme from Sigma-Aldrich. Triplicates were performed for each sample set (colony 1 culture, colony 2 culture, colony 1 lysate, and colony 2 lysate).

The galactose induced sample shows greater reduction in OD, followed by the sample with no inducer or repressor (sucrose), and the glucose repressed sample with the least amount of reduction in OD. However, since the change in OD occurs on such a small scale (<math>delta</math>OD ~ 0.05), the differences between the samples may not be significant, especially when the positive control, 0.1 g/L chicken lysozyme from Sigma, shows a 5-6 time greater reduction in OD (<math>delta</math>OD ~ 0.25) than the galactose induced sample. Nevertheless, the lysozyme is indeed active, despite its very low activity. Performance optimization needs to be done to establish the optimal temperature and pH for the lysozyme. Because there are such high fluctuations in the OD trend, we decided to conduct a longer, 12 hour assay to establish a more stable trend.

For each sample set in the above figure, there was an induced (galactose), inhibited (glucose), and unaffected neutral sample (sucrose). Overall, all of the samples exhibited a decrease in OD450, implying that CLYC was present in all 12 of the samples. However, according to the data, the Gal1 promoter does not function predictably. In some instances, glucose does not inhibit the expression of CLYC and in other instances galactose does not induce the expression of CLYC (See figures 4 and 5). Thus, although we were able to observe the detection of CLYC, we were not able to effectively suppress or induce its expression with the Gal1 promoter.

Conclusion

When analyzing the results from the agar diffusion assay, we can conclude that our CLYC plasmid is ineffective at producing a significant amount of functional lysozyme. However, we only tested samples from our cultures, rather than trying to purify out any lysozyme produced and then testing for functionality. As shown in a past study conducted with lysozyme secreting Pichia Pastoris, the highest yield of purified lysozyme was only 0.4 mg/mL (Masuda et al). This example shows that the expression levels of lysozyme in yeast may simply be too low on its own in order to produce a significant concentration. Due to this fact, our samples were likely too diluted to visually display the same type of functionality as our controls. In the future, further testing may include the purification of any lysozyme produced, followed by an agar diffusion assay. While we can conclude the lack of significant lysozyme production, the agar diffusion results cannot exclude the possibility of any lysozyme production whatsoever. Our CLYC plasmid does not produce the qualitative results that we would like to see, but it could still be successful in producing lysozyme to some extent. In order to detect any sort of lysozyme production, we ran kinetic OD assays in order to detect small amounts of bacterial lysis. First, we ran a short-term kinetic OD assay over a 45 minute time period. Based on the results, it can be concluded that that in the short term, the CLYC lysozyme is not nearly as active as the 0.1 g/L hen egg-white lysozyme. However, the data does demonstrate that the GAL1 promoter is working to some extent. The induced samples display a greater ΔOD than either the neutral samples or inhibited samples. And while the difference is not all that large (within 0.005 AU of one another), the p-values clearly indicate that the induced samples are significant when compared to the neutral samples, which naturally lends to it being significant from the inhibited samples as well. The neutral samples and inhibited samples dataset are insignificant, which can be due to the fact that the GAL1 promoter may not repressed completely by glucose, evident by the miniscule amount of activity that is present. To address this issue, the dynamic range of repression would need to be established through more experimentation to find the upper limit of repression. Clearly, 0.2% glucose is not enough to completely repress the promoter. Nonetheless, the conclusion still holds that the induced LYZ gene is producing lysozyme. The short-term kinetic OD assay may have displayed lysozyme production, but there was a bit of fluctuation in our OD readings. In order to obtain a clearer OD trend and detect lysozyme production over a longer period of time, we decided to run the same kinetic OD assay, but for a much longer time period, specifically 12 hours. Our results for the long term assay displayed the same results as the short term, but with less fluctuation in OD readings. Because the protein titer from the yeast appears to be very low, the next step would be to isolate the protein, purify, and concentrate it before running any more assays. Additionally, temperature and pH ranges need to be optimized for better lysozyme performance. Finally, the yeast expression host can be also be optimized for lysozyme expression, deleting any genes that may inhibit the expression of the CLYC vector in yeast. This deleted strain will be the workhorse for lysozyme production. The agar diffusion and kinetic OD assays will be repeated using the purified concentrated lysozyme instead of directly inoculating with low-expressing transformants. To further test the application of a lysozyme-secreting yeast strain in fermentation decontamination, an application assay that can be used to simulate fermentation conditions is a fermentation assay. In this assay, we would measure turbidity, lactic acid concentration and pH by growing up the optimized expression host in high sugar YPD + 16% sucrose media (YPD16S) inoculated with lactic acid bacteria, the main contaminant in fermentation broth. If the lysozyme secreted by the yeast is well behaved, the turbidity of the broth should decrease over time and less lactic acid concentration would be detected. Finally, because lysozyme’s turnover number is known to be very low, we can test synergistic interactions between lysozyme and other antibacterial agents. Overall, our project demonstrates that lysozyme can successfully be produced in yeast. Furthermore, the results from our Kinetic OD assays demonstrate that said lysozyme can be functional in lysing Gram-positive bacteria as well. On the other hand, our device is flawed. The amount of lysozyme produced is not significant enough to have the impact needed to effectively decontaminate brews during the fermentation process. In the end, further research needs to be done on whether or not the expression levels of yeast and other organisms used in microbial food cultures is significant enough for decontamination.

Stanford Location

Glycerol Stock Information: Barcode #: 0133023900; Plasmid name: Chicken Lysozyme in Yeast Chimera; Antibiotic Resistance: Kanamycin; DNA 2.0 Gene ID: 193520; Organism expressed in: S. cerevisiae; Sensor/Actuator: Actuator - chicken lysozyme

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