We got the 3D prints of our test manometer today. This is what it looked like:
We will be reprinting the manometer as one single piece in order to use it for next class.
Todays focus was on our wax paper design. We printed a smaller starburst and placed the solid YPD onto the different channels. We kept the environment sterile with an ethanol burner and a UV light.
Then, to contain the yeast in a sterile environment as it grows, we placed the paper in a petri dish.
We noticed from the presentations of other groups that people were having an issue with their yeast drying out because they need to maintain moisture in their environments. To help this, we pipetted distilled water into the spaces between the channels. We hope to observe growth in our yeast next class without it drying out. We incubated the plate at 30 degrees Celsius.
During last lab period we ordered a 3D print of a test mini-manometer and a box for it. Today we only received the box because Morgan canceled the rest of our prints due to the walls being too thin and the box breaking. We enlarged the dimensions of the prints (the borders of the larger box were increased from .5 mm to 5mm and the borders for the smaller box were increased by from .5mm to 2mm) and expect to receive them by next class period.
In respect to the second test – the test of yeast growth on paper – the parafilm barrier worked perfectly in regards to keeping the YPD from going through the paper as shown before.
However, we decided to cut each of the branches of the starburst separately because there is no barrier in the filter paper that would stop the YPD from each pH (branch) from mixing with each other, which would ruin our idea of keeping a neutral center. This was due to the fact that our YPD was very liquid.
We encountered a few issues regarding the making of solid media, which delayed us in the process of laying the media on the respective branches and stopping it from moving into other branches. However, we were able to fix the viscosity of our YPD by microwaving it for 30 seconds and so we realized that we no longer need to cut and separate the different branches from each other.
Today we will be printing a test mini-manometer, it will be printed in three pieces. The tube, the square well, and the holding box for the PDMS. To eat away at the 3D printed model MEK (Methylethylkeytone or Butanone) or hair and grease drain cleaner depending on price.
Researcher, creating 3D model on tinker-CAD.
well boxPDMS holder (printed horizontally for printer
For our 2D design, we are considering different methods of sterilization for the parafilm and filter paper based design we created. UV sterilization and ethylene oxide are currently being considered to sterilize the filter paper before parafilm attachment ((melted based onSimple and rapid fabrication of paper microfluidic devices utilizing Parafilm by Nathalie Horner)). The parafilm barrier was successful however the wax print needs a barrier to prevent running into the starburst’s branches. We will be trying a new method where the arms are physically separated then reattached when gel is solidified.
Today we had our first presentation in the Chemistry section of the lab. We uploaded our 3D model to the class Box. We also created a mass batch of YPD, using 20 grams of dextrose, 20 grams of peptone, and 10 grams of Yeast Extract, filling up the rest of the bottle to 1 Liter with distilled water and sent it to be autoclaved. We also printed out our wax paper assay and put it in a hot plate set at 100 degrees Celsius to melt the wax through to the other side of the paper. After about an hour, we removed the wax assay from the hot plate to check how much the wax melted through. We noticed that the wax did not melt all the way through towards the ends, so we stuck it in an oven. However, there was a problem with the design in that some wells in the starburst had wider openings than others. We’ll try to fix it, but if it’s impossible, we’ll just roll with it.
We decided to wait until next class before adjusting the pH in the autoclaved YPD solution due to time constraints. We also researched how we would dissolve the PLA filaments from the 3D Printer so that the PDMS would be the only thing left. We’ll either use something called MEK (methyl ethyl keystone) or Hair and Grease Remover. We could also use ethylene oxide; we are still debating what we are going to do. Also, we continued to modify our 3D Design before sending it off to the Printer. We’ll meet with our connection in the Media Lab in order to confirm our design is viable. That’s all folks!
Today we are designing the 2D model for our wax paper assay and the 3D model for our PDSM assay.
The 2D model was designed on illustrator:
On the wax paper assay will have have one star for each strain of yeast (Wild Type, SAK1, and TOS3 D) with three replicates each for a total of 9 stars.
The 3D model was designed on Tinkercad. We had to take in account the output rate of CO2 in metabolizing yeast at 30°C to decide what diameter and height of the channel of the measuring end of the manometer should be. The goal is to find a optimum diameter and height where the manometer can be read easily but also the manometer remains functional. We decided to work with the dimensions of 1.8mm in diameter and 70mm in height. If the reservoir for the yeast of our model is completely saturated and the experiment ran for 70 minutes then that would make the manometer theoretically move 7000cm given diameter, but if we bring yeast concentration down a magnitude of 10^(-2) from complete saturation, then manometer will work within the space we have.
Today is the first day of the Chemistry Lab. We must recreate the Yeast (Saccharomyces Cerevisiae) experiment, but this time we plan on creating, through 3D printing, multiple open-ended manometers that will measure metabolic rates of anaerobic fermentation. Inside the container there will be the inoculated YPD (Wild type, TOS3 Deficient, or SAK1 Deficient) it will metabolize and produce carbon dioxide as a byproduct.Ethanol will also be placed in the manometer and will settle. The air pressure (carbon dioxide) will force the ethanol out of its initial position and then we can measure the change of air pressure. This information will let us measure metabolic rate over a certain period of time. We also plan on solidifying the gel with the appropriate pH in the wax paper. The wax paper will be designed in a star-shape. With the inoculated YPD in the center and the pH levels branched out from the center. The growth of the yeast will differ depending on the pH level branch (2-9 increments of 1). That is our plan.