Team:Newcastle/InterLab
Alternative Roots
InterLab Study
Newcastle InterLab Study
Overview
Reproducibility, the ability to carry out and replicate the results of a single experiment, is an important aspect of scientific disciplines. However, in the life sciences, the concept of reproducibility has become a large problem. A vast number of experiments throughout the various disciplines of the life sciences are seen to lack a reproducible nature, ultimately costing and inadvertently wasting large sums of money. Synthetic biology is no exception to the troubles of reproducibility, with inaccurate part characterisation impacting the the ability to use Bio-Design Automation (BDA) to build fully functioning, novel synthetic gene circuits.
iGEM devised the inter-lab study – an annual, large-scale study carried out by institutions around the world by researchers of varying experience levels – to determine the reproducibility of individual synthetic biology protocols, assessing and addressing the limiting factors of reproducibility. The study attempted to address variation between different models of plate readers by producing a step by step protocol for measurement and analysis, allowing the production of directly comparable fluorescence measurements. In addition, ribosome binding site (RBS) sequences designed to increase precision of expression were included in the devices to be transformed into the host cells.
A weakness in the measurement of fluorescence relative to OD600, as with previous IGEM interlab protocols, is the potential discrepancy between optical density and actual cell concentration. This year the IGEM study aims to reduce lab-to-lab variability further by measuring GFP fluorescence relative to absolute cell counts or colony forming units. Normalisation of fluorescence to colony forming units goes further by allowing measurement of fluorescence relative only to viable cells, and thus a more accurate measurement of promoter strength, whereas OD600 and absolute cell count measures cannot differentiate between viable and non-viable cells.
Stage One
Design
Once the project idea was finalised, the team began looking for cheap, efficient and standardised methods for growing plants in iGEM. The hope was that such an item existed that would meet these specifications as well as being a closed container to prevent contamination and also providing a high throughput of plants. It was soon established that such an item did not exist to meet our specifications. Therefore, to combat this issue, it was decided that the best way forward would be to design our own hydroponics system. This would allow us to grow large amounts of Arabidopsis in a controlled setting for the purposes of our project. Several team members were assigned to this “sub-project”.
Before getting hands-on in building the system, the team as a whole established a few design parameters. For example, the system needed to be cheap and easy to build from scratch. This is so future iGEM teams are able to construct the system for their own needs and even build upon our design, as necessary. Additionally, the system must be versatile, open-source and easily adapted for various conditions such as light intensity and wavelength. By adopting such an open and adaptable design the intention is that the end-user is able to effortlessly match the system to their needs, without getting entangled in streams of code.
Several weeks were spent modifying the design until a design was found that met all the above criteria, the specifications of the design can be seen below.
UP TO
SEEDS CAN BE GROWN
IN HYDROPONICS
APPROXIMATELY
KWH OF POWER ANNUALLY
USED TO POWER SYSTEM
PROVIDES UP TO
LUX OF LIGHT
TO GROW SEEDS
CONTAINS
INDIVIDUALLY ADDRESSABLE
LOW-POWER LED'S
Stage Two
Assemble
Having identified the design parameters for the system, the next stage was to begin ordering parts and putting it together. The system was divided into three independent, functional sub-systems to make the task of assembling the system more manageable and allowing team members to focus on the sub-system that most suited their specialty. These three sub-systems were hardware, software and biological aspects.
The function of the hardware is to contain the electronics and organisms, power the LED’s/microcontroller and maximise the light available to the plants. Containment is through the use of a sealed box, with a detachable lid for access. This box is glued with tin foil and sprayed black to minimise exchange of light with the environment. Powering the LED’s proved to be more difficult, taking our engineers many days to find the optimal solution. You can find all the grizzly details on this process here. However, essentially the system is powered from a 5V 2.1A AC adapter that plugs straight in to your mains power supply. Alternatively, you can use 4 AA batteries to power the system for short periods of time if necessary. The LED’s are wired in parallel so the same light is provided along the length of the container. This can be seen from images in the Gallery.
The purpose of the software is to control the LED’s, by allowing the user to easily adapt features such as light intensity, wavelength and also specify the length of the day/night cycle. For our design, we use the Arduino UNO microcontroller to control these characteristics as it offers a user-friendly interface and is well-suited to our design. You can find all the code laid bare and a guide to the Arduino here.
The engineers, hard at work trying to troubleshoot issues with the system.
The finished product, set to a rainbow function that cycles through various wavelengths of light
Stage Three
Test
Substantial time was spent carrying out extensive research, both inside and outside the lab, in order to optimise the system for the target audience. This included speaking with organisations and individuals in industry who are involved with hydroponics-based systems or those who may be interested in working with such a system in the future. Some of the individuals we liaised with include Chris Tapsell, the Research Director of KWS UK, one of the biggest seed companies in the world, and Richard Ballard, co-founder of Growing Underground in London where they hydroponically grow micro greens and salad leaves 33 metres below the ground. These potential clients helped us focus our product so that it can better meet the needs of our clients.In addition to gathering external opinion on our system, we also did our own tests on system performance. This included tests to verify the optimal light intensity, wavelength and positioning. The graph below illustrates how the light intensity (measured in lux) varies over time (in seconds) when the system is operated under various wavelengths of light. The black line indicates the system running with the rainbow function loaded which cyclically varies the light wavelength. As the results showed that blue, red and purple light and provided the most lux we are currently using these in the system but plan to use the rainbow function too in future to see how this affects growth or the aesthetics of the plant.
Hardware
Gallery
