Hardware

1. The Problem

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.

Other machines simply lack the capabilities to ...

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 manufactured, tested and fully documented.

They are:

GCODe Pro

And

GCODe Mini

Please follow the links to find out about their capabilities, design, cost and assembly instructions, and the results of our tests on them.

2. 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.

Design

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.

[diagram]

[pictures]

Assembly Instructions

GCODe Mini Assembly

Software

Gallery

3. GCODe

Intro

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 has the same analysis unit as the Mini, but uses a network of servos and a peristaltic pump to draw precise amounts of culture and dilute it to any desired level before measurement. Optical Density is measured by drawing the liquid into a standard cuvette, which allows the flexibility to grow unlimited amounts of culture in a conical flask or any other vessel.

Design

THe GCODE Pro has X main components

THe Analysis unit uses a cuvette supplied and drained by a needle, but the measurement system is the same as the one used in the Mini

The Sampling Unit draws liquid from the conical flask, or culture vessel, dilutes it as programmed, and delivers the culture to the cuvette. THe pipes are cleaned between every run by washing with ethanol.

Assembly Instructions

Software

Feedback: user interface was not usable by non coders, switched to html interface and gui

Gallery

4. Results

Calibration

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, with the optical density measured every three hours with GCODe and an ELISA plate-reader.

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 !

The normalized GCODe reading is

Assembly from Instructions

In order to test whether our written instructions, images and design files were clear and complete enough to actually allow biologists to assemble our device, we invited first year undergrads from our university to test them out. We provided them with parts (since that would have been too much to ask) , and the instructions you see on our wiki. In less than two hours, they successfully assembled the GCODe Mini!

[insert pictures of beaming UGs]

Inspirations and Future Plans

• Our project builds on the work of the 2014 Aachen team, but improves on their work in several ways

• We added an LED current driver, which is vital to maintaining a steady illumination in countries with fluctuating power supply voltage
• Our lab-scale (as opposed to micro-scale device allows the use of large quantities of culture, and via dilution and flushing, can work with organisms like yeast that would otherwise clog the pipes.

….

GCODe can be applied to bacterial systems, yeast and other fungi, and any microbes whose growth can be measured by OD. It can be used to run complete growth curves and produce graphs without any human intervention, and can alert you when your cells reach the stage of growth that you want.

Improvements we want to make

• depends on what version/stage we actually finish
• add reagents
• miniaturize
• flask unit

• maintain code/ make app
• market prefab version

Testimonials

Highlights from our Hardware Journal

• Our design evolved greatly over the course of the months we spent working on it. Initially we planned to simply have the culture growing in a test tube, but our wetlab team was working with yeast at that point, which grows to ODs far greater than anything that can be measured without dilution. Our teammates suggested that dilution would be extremely useful in expanding the utility of ABODE beyond E.Coli .
• for each bullet, have a nice sounding quote from the hardware journal

• We also realised that it was necessary to make our device compatible with conical flasks, because those are what biologists use when they need to make large amounts of culture for any purpose apart from measing its rate of growth.
• Our initial design had a special 3d printed test tube holder, but tests in the IISc undergraduate lab showed that even very similar looking 'standard' test tubes have differing bore sizes. We then switched to a much simpler and cheaper holder made of laser-cut acrylic that allows many test tubes to be fit in it, with a bit of insulating tape.
• We initally planned to use an elegant seven-sided connector \\insert picture to link our pipes, but then we realised that the 3d printing plastic it was made of was not compatible with the ethanol we use to sterilize our system. We replaced it with medical-grade biocompatible 3-way stopcocks.
• The Doomed Multiplexer \\put pics

• we struggled for a long time, inventing ever more complicated ways of closing off 5 pipes while opening the last - using just one programmable motor. What stymied us was surprising - humans are strong. Pinching a pipe closed is no easy task for a cheap little motor. Eventually we switched to a system of one servo per pipe connection, which took up much more space but offered finer grained control and a lower chance of catastrophic plumbing failure.

• Our initial user interface was rather hard to use, so we came up with a web-based system, since we didnt want to force users to download an application.
• Something that worried us a lot was that our readings just didn't match the spectrophotometer readings. We talked to a professor who worked in optical systems and microfluidics, and he pointed out that the coefficient of absorbance in Beer-Lamberts law is different for every optical system, and so there was no need to worry if the readings were proportional.
• notifications at a particular od, respnse to feedback from Srinath, UG instructor //pic of pushbullet screenshot
• Sai Siva gorthi

• Pointed out that epsilon in different systems is different, so we needn't worry about getting different readings from the spectrophotometer as long as they were proportional

150 Words for Special Prize

Does the hardware address a need or problem in synthetic biology?Did the team conduct user testing and learn from user feedback?Did the team demonstrate utility and functionality in their hardware proof of concept?Is the documentation of the hardware system sufficient to enable reproduction by other teams?

We built a fully automated microbial growth management system that eliminates the need to take growth curves and measure optical densities manually, with the capability to draw a sample, dilute it and measure the OD, while maintaining a sterile environment. We also designed software that allows GCODe to take readings at pre-set intervals, plot graphs and notify experimentalists as desired. Our incremental progress, evolving based on the needs and suggestions of the biologists around us, has resulted in two models, the GCODe Mini and GCODe Pro, both of which have been successfully tested in a laboratory setting. The Mini has also been assembled by undergraduates working only from our instructions as seen on the wiki. GCODe is the low-cost open source solution that finally removes the pain from growth curves - across the globe.