Part:BBa_M36763:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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.

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

User Reviews

UNIQ3fda40c506b62e0f-partinfo-00000000-QINU UNIQ3fda40c506b62e0f-partinfo-00000001-QINU