We collaborated with other iGEM teams in three different ways-
- Wet lab work for team IISc Bangalore: Data collection for machine learning
- All India iGEM Meet-up at IISER Pune
- 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
- 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!
- Incubate the culture at 30⁰C,overnight, at 180 RPM.
DAY 2: The Growth Curvesa)MAKING THE SECONDARY INOCULUM
- Add 2.5 mL of primary inoculum to 247.5 mL of YPD/YPAD in a 1L flask in sterile environment.
- Swirl flask gently.
- Take‘0’th time point sample for both replicates and store it at 4 degrees.
- Seal mouth of flask with a cotton plug, incubate at 30⁰C and 180 RPM.
- Take samples every one hour for the growth curve for 16 hours.(Comment-We literally spent the entire night in the lab)
- Resuspending Culture
- For each time point, pipette 1 mL of culture into an eppendorf in a sterile environment.
- Centrifuge aliquot at 1500 RPM (or 2000g) for 2 mins to pellet cells.
- In sterile environment, remove the supernatant and resuspend cells in 1 mL PB
- Spin down at 1500rpm for 2mins.
- Resuspend the cells in 4% PFA
- Incubate at RT for 90mins
- Spin down at 1500rpm for 2mins
- Resuspend in 1mL PBS
- Make duplicates of 200 &muL PBS + 200 &muL culture
- Add 10 &muL of safranin to one and 10 &muL of methylene blue to the other.
- Vortex gently and let solution sit for 3 - 5 minutes (max) at room temperature.
- Load and visualize all of the above samples.
- Hemocytometer: Loading
- Clean hemocytometer surface and coverslip with 70% ethanol.
- Place coverslip on hemocytometer.
- Then, take 10 uL of sample and load it onto the hemocytometer carefully as such:
- Invert the test tube several times to resuspend the cells.
- Pipette 10 uL of sample at once with the smallest micropipette tip available.
- Add the 10 uL of sample to the V-shaped groove in the hemocytometer.
- Hemocytometer: Visualizing
- Let the sample rest on the hemocytometer for 2 mins before viewing under microscope. If cells are on a different focal plane from the hemocytometer, let the cells rest longeror load lesser volumeof sample.
- View under 10x magnification, center central grid of hemocytometer under objective. Capture image of central grid with the help of phone camera
- View under 40x magnification, take images of grid locations 1,2,3,4 and 5.
Order of pictures taken for each sample:
- 10x-whole grid
- Top left grid
- Top right grid
- Central grid
- Bottom left grid
- Bottom right grid
(ii) All India iGEM meet-up 2017
- This collaborative event was a huge success. 9 out the the 10 Indian iGEM teams(including IISER Pune) joined us in our Institute for a Mini-Jamboree. Team Peshawar Pakistan also joined us via Skype. The meet-up was 2 days of intense discussions and talks on various projects. Teams helped each other by posing potential problems with the projects and then brainstorming solutions for the same. We talked about safety, viability, usefulness of each project. This helped us a lot in improving our work. We helped team Peshawar in selecting a topic for the project by discussing pros and cons of each topic.
(iii) Social media and whatsapp groups
- We got to talk to many teams via our facebook page. We filled survey forms of many teams and also forwarded them to the others.
- We had a very active iGEM India whatsapp group which was extremely helpful throughout the course of the project. Be it the Visa application or DNA ordering or troubleshoot of some experiment, this group always been of incalculable help.