Team:IISER-Pune-India/Collaborations

Medal Criteria

Collaborations

“Many ideas grow better when transplanted into another mind than the one where they sprang up.”-Oliver Wendell Holmes

We collaborated with other iGEM teams in three different ways-

  1. Wet lab work for team IISc Bangalore: Data collection for machine learning
  2. All India iGEM Meet-up at IISER Pune
  3. iGEM groups on social media and Whatsapp

IISc Bangalore Collaboration

Their team was trying to build a precise and time effective way to measure the cell count of yeast cultures. They wanted to collect a huge amount of data to train a ​machine learning algorithm that they had designed for counting these cells. Collecting such huge amount of data means growing hundreds of yeast cultures,collecting samples every hour and then imaging it under the microscope on hemocytometer. It is a tedious task to be done alone by a team. Thus, with this collaboration our team was expected to generate images for the algorithm to analyze by monitoring one growth curve for Saccharomyces cerevisiae.

An introduction to machine learning that team IISc Bangalore has designed can be found here

We were asked to follow the following protocol-

Mentioned below is the protocol that we followed in which we have made certain modifications to the above protocol-

DAY 1: Primary Inoculum

  1. In a sterile hood, inoculate a colony of ​S.cerevisiae ​in ​5 mL ​of ​YPD/YPAD​( 2 replicates). (Since we didn’t have the lab strain of ​S.cerevisiae and this experiment needed only quantitative data for the machine learning algorithm ,we used common baker’s yeast for the culture.) Comment-This was something exciting!
  2. Incubate the culture at ​30⁰C,​​overnight​, at ​180 RPM​.

DAY 2: The Growth Curves

a) MAKING THE SECONDARY INOCULUM

  1. Add 2.5 mL of primary inoculum to 247.5 mL of YPD/YPAD in a 1L flask in sterile environment.
  2. Swirl flask gently.
  3. Take​‘0’th time point ​sample for both replicates and store it at 4 degrees.
  4. Seal mouth of flask with a cotton plug, incubate at ​30⁰C ​and ​180 RPM​.
  5. Take samples ​every one hour ​for the growth curve for ​16 hours​.(Comment-We literally spent the entire night in the lab)

b) TAKING MEASUREMENTS

  • i)Resuspending Culture
  1. For each time point, pipette ​1 mL ​of culture into an eppendorf in a ​sterile environment​.
  2. Centrifuge aliquot at ​1500 RPM (or 2000g) ​for ​2 mins ​to pellet cells.
  3. In sterile environment, remove the supernatant and resuspend cells in 1 mL PB
  4. Spin down at 1500rpm for 2mins.
  5. Resuspend the cells in 4% PFA
  6. Incubate at RT for 90mins
  7. Spin down at 1500rpm for 2mins
  8. Resuspend in 1mL PBS

  • ii) Staining
    1. Make duplicates of 200 uL PBS + 200 uL culture
    2. Add 10 uL of safranin to one and 10 uL of methylene blue to the other.
    3. Vortex gently and let solution sit for 3 - 5 minutes (​max​) at room temperature.
    4. Load and visualize all of the above samples.