Part:BBa_M50447:Experience

Negative functionality.

Methods:

Our DNA construct was synthesized by Atum. Once received, we transformed this construct into yeast using a previously established protocol using -URA3 as a selection marker in yeast extract-peptone-dextrose (YPD) media.19 After 48 hour growth at 30℃, we isolated a single clone and streaked it onto a new YPD plate with the same -URA3 selection. This single yeast clone was used to create liquid cultures for all subsequent experimentation.

Glucose Induction Experiment

Yeast liquid cultures from our previously prepared single clone were grown overnight in 2% raffinose. Absorbance measurements at 600 nm were taken following the overnight incubation. Cells were diluted to an optical density of 0.1 in YPD media and mixed with glucose to start the induction. Because we wanted to maintain a constant carbon source concentration of 2%, we added raffinose as we varied glucose. Seventeen concentrations of glucose were tested ranging from 0 mM (0%) to 111 mM (2%). Increments of 5 mM were used for the range from 0 mM - 50 mM and increments of 10 mM were used from 60 mM - 111 mM. Five separate controls were also run. This included three blanks; one with just YPD, one with YPD plus glucose as the carbon source, and one with YPD plus the identified ideal non-glucose carbon source; one control with cells (0.1 O.D.) but no carbon source; and one sample with cells taken from the previous liquid culture. 200 μL of each prepared induction mix was added to a separate well in a clear bottom 96-well plate. Each induction mix was run in triplicate on the plate and incubated for 24 hours. The plate was incubated on a shaker inside the plate reader at 200 RPM and temperature controlled at 30℃. GFP expression was quantified with two different fluorescent reads on the plate reader, one with an excitation/emission of 480 nm / 520 nm and one with an excitation/emission of 395 nm / 509 nm. Fluorescent reads were taken every 30 minutes during the 24 hour induction. Absorbance at 600 nm was also measured every 30 minutes with the fluorescent reads to monitor cell growth.

Western Blot

The protocol for the Western Blot was obtained from our laboratory manual.19 We used a rabbit anti-His tag antibody directed against the His-tag on our insulin protein as a primary antibody (1:1000 dilution) and used a goat anti-rabbit secondary antibody (1:1000 dilution) as our secondary antibody. The secondary antibody was conjugated to HRP, which allowed us to visualize protein production after adding Opti-4CN chromogenic substrate.

Results

We knew that our plasmid sequence was correct because it was sequence verified by Atum before it was sent out. From here, the goal of our initial prep experiments was to confirm that our plasmid had actually entered our yeast cells, in other words to confirm that our transformation had worked correctly. Our experimental plate, which was treated with kanamycin, from the transformation showed colonies after a 48 hour incubation at 30℃ whereas no colonies appeared on our negative control. Furthermore, a single isolated colony grew up properly when it was streaked onto a new plate with kanamycin, which showed that our transformation was successful. Once we were certain that our plasmid had properly entered our yeast cells and was the correct sequence, we proceeded to perform our 24 hour glucose induction experiment to measure the effect of changing glucose concentration on insulin production. We measured change in insulin production via GFP expression since EGFP was linked to the insulin gene via a T2A peptide under the same promoter. Yeast cells were grown in 2% raffinose overnight and then induced with varying levels of glucose ranging from 0 mM - 111 mM (2%).

We did not see the results we wanted to see. GFP expression decreased over time and did not vary with glucose concentration. Both "induced" and uninduced cells decreased at the same rate, suggesting that glucose exerted no role in making our promoter express GFP.

see figure 2.

Scatter plot showing GFP expression over time with different concentrations of glucose induction. Glucose induction at concentrations of 0, 25, 50, 80, 100, and 110 mM are shown along with additional controls. Time is shown on the x-axis and GFP expression in relative fluorescence units is shown on the y-axis. An excitation filter of 480 nm with an emission filter of 520 nm was used for the data in this graph. Black data points represent glucose induced samples and colored data points represent controls. Error bars are +/- S.D.

Western blot - we saw no increase in insulin production at high concentrations of glucose (2%) vs a negative contorl with 0% glucose.

see figure 3.

Western blot image for insulin protein in induced and non-induced yeast cells. A 10 kDa prestained protein ladder was run in the first lane, cell lysates from induced yeast was run in the second lane, and cell lysates from non-induced yeast cells was run in the third lane. Induced yeast were cultured overnight in 2% glucose + YPD and non-induced cells were cultured overnight in 2% raffinose + YPD.