Team:IISc-Bangalore/Hardware

  1. The Challenge
  2. GCODe Mini
  3. GCODe Pro
  4. Results

The Challenge

Measuring the growth of cells is a vital task in any biology lab across the world. Thousands of dollars are spent on spectrophotometers and analysers to measure the optical densities of cell cultures. More importantly, hundreds of researcher-hours are spent taking samples, diluting them and measuring the optical density, again and again, through the long nights of running growth curves.

There do exist automatic devices that can measure OD, such as the Bioscreen C, but their prohibitive cost (in the tens of thousands of dollars), low volume capacity, and inability to handle high ODs make them non-viable as a substitute for manual pipetting in most experimental scenarios.

There has to be a better way. That way is GCODe.

GCODe (Growth Curve and Optical DEnsity) is an optical density measurement device that will not only take readings at pre-programmed intervals or continuously, but will also aerate and dilute the culture as required. It can even send you a message when the OD reaches a particular level, just in time to you to start the next stage of your experiment. Fundamentally, it automates the grunt work of growth curves, in a manner that allows you to walk away from the lab, secure in the knowledge that you will be alerted when your cells are ready for you.

Our work stemmed from our own frustrations running growth curves night after night, and was also inspired by the OD meter built by the 2014 Aachen iGEM team. We worked incrementally, starting with a rudimentary box of wiring, designing version after version in response to feedback from the iGEM wetlab team, as they trialled our device, and other labs and professors from across IISc.

We currently have two versions of GCODe - The GCODE Mini and the GCODe Pro. The GCODe Mini has been developed, fabricated, tested and fully documented. The GCODe Pro is still under development and is in its third version.

GCODe Mini

Intro

The GCODe Mini is a sleek black acrylic cuboid that houses a test tube that will hold your culture. Connect the Mini to your laptop via USB, tell it what to do with our associated software, place it in the shaker-incubator, press Start … aaand you’re done !

The Mini can:

Take readings of the optical density at specified intervals for as long as required. Alert you via pushbullet notifications when the OD increases by a specified amount, or at regular time intervals, or when the OD reaches a desired value. Plot a graph of OD vs time.

Description

The GCODe Mini has two components, the Power Unit and the Analysis Unit. The Analysis Unit contains the Arduino, and the test tube. It will be connected to the laptop, and goes inside the shaker. The shaker should be plugged into the Power unit (which requires Mains power), so that it can be turned off during measurement to give a stable reading.

How it Works

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GCODe Pro

Intro

Although the GCODe Mini is a great little device, and can make life much simpler in a large range of situations, it has its limitations. The GCODe Pro was designed to overcome these limitations.

While the Mini needed the culture to be grown in a test tube, the GCODe Pro works perfectly with the large quantities of a standard conical flask. It will automatically do dilution for you, which means it can measure an almost unlimited range of ODs, including those that occur in yeast and other organisms not compatible with regular plate-readers. The GCODe Pro uses a network of servos and a peristaltic pump to draw precise amounts of culture and dilute it to any desired level before transferring it to the Analysis Unit, which then measures Optical Density by drawing the liquid into a standard cuvette. This allows the flexibility to grow unlimited amounts of culture in a conical flask or any other vessel. The GCODe Pro is an interplay of fluidics, optics, and electronics.

How it Works

Like the GCODe Mini, the GCODe Pro takes OD measurements at regular intervals of time to automate a Growth Curve. During one cycle of measurement, the following processes happen automatically:

  1. The shaker stops shaking
  2. A certain volume of sample is drawn from the flask containing the culture.
  3. This sample is then diluted according to experimental needs.
  4. The diluted sample is mixed by resuspension (pumping out and in).
  5. The diluted sample is transferred to the Analysis Unit.
  6. The OD of the liquid in the Analysis Unit is measured.
  7. The Sample is then discarded into a discard container.
  8. The tubing containing excess sample undergoes a cleaning process to maintain sterility.
  9. The shaker resumes shaking.

The GCODe Pro comprises of 5 different “units” to help do this. They are the:

  1. Power Unit
  2. Sampling Unit
  3. Mixing Unit
  4. Flask Unit
  5. Analysis Unit

The following flowchart shows a schematic representation of fluid flow in the Pro. [IMAGE]

Power Unit

The power unit is responsible for turning the shaker OFF during measurement, and ON when it is not measuring.

Sampling Unit

The Sampling Unit handles the fluidics. It is responsible for all the fluid transfer processes that happen during measurement. Fluid flow is controlled by a peristaltic pump and several servo motors connected to one stopcock each.

Mixing Unit

The mixing unit is a container in which the Sampling Unit mixes and dilutes fluids. It also acts as a buffer for fluid transfer.

Flask Unit

The flask unit is the part which goes inside the flask in the shaker. It contains the tubing to draw the sample from the flask. It also contains the tubing required for the cleaning mechanism.

Analysis Unit

This is where the actual OD measurement happens. It consists of an LED-photodiode setup similar to the one on the Mini.

Results

Growth Curves

As soon as the GCODe Mini was assembled and calibrated, we put it to work. To find out whether GCODe could actually measure optical densities in a reproducible manner, we tested it in Prof. Dipshikha Chakravortty’s lab under the supervision of Jeswin Joseph, her PhD student.

Our growth curves used Salmonella typhi, E.Coli with the optical density measured every three hours with GCODe and an ELISA plate-reader.

[[File:T--IISc-Bangalore--hardware_gc1.jpg|600px]]

Salmonella Growth curve on Date1

[[File:T--IISc-Bangalore--hardware_gc2.jpg|600px]]

Salmonella Growth curve on Date2

The dark blue line is the OD measured by GCODe, and the red line is the OD as measured at 600nm with a spectrophotometer. While the absolute values of the OD differ, due to the different wavelengths of the LEDs, normalizing with the ratio of ODs at 12 hours (green line) or 24 hours (cyan) shows near-perfect agreement !

[[File:T--IISc-Bangalore--hardware_gc3.png|600px]]

Salmonella Growth curve on Date3

We also ran Growth curves on the Mini with E.Coli [[File:T--IISc-Bangalore--ecoli1.jpg|600px]]

E. Coli Growth curve on Date2

[[File:T--IISc-Bangalore--ecoli2.jpg|600px]]

E. Coli Growth curve on Date2

Notice that the readings from the Mini form an even smooother curve than those from the spectrophotometer! This is probably because the GCODe readings were taken instantaneously, while it takes time to load the cuvette into the spectrophotometer and take a reading, allowing lots more opportunities for human error.

Assembly from Instructions

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Cost

pretty low

Testimonials

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[[File:T--IISc-Bangalore--logo-new.png|300px]]

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