Team:UNOTT/Design3






Key. coli Transport Device Design


The lock and the key: how does it work?

A lock would be comprised of a fluorescence detection device which can only be accessed when the fluorescence profile of the ‘key’ matches the reference fluorescence profile of the ‘lock’. An image recognition software, designed by one of the Nottingham researchers, compares the two spectra for likeness and allows access only when the correct spectrum ratios have been obtained.

Therefore, the fluorescent patterns of the key and reference samples need to have minimal variance to allow correct authentication. If otherwise, the accuracy and completeness of emission signals is invalidated and fewer discernible combinations are possible.

This is where the requirement for a key transport device came in, that would allow survival of the bacteria, revival under controlled conditions and easy measurement of the expression signal. This device can be as small as a conventional door lock.

Additionally, we are developing an accompanying Key. coli modelling software which could predict fluorescence outcomes under all possible conditions and allow each key to be categorised separately by its output.

Cell storage



Freeze drying, the golden standard for the long-term storage of microorganisms1, was the method of choice for storage of the Key. coli cells.

Key. coli cells will be freeze-dried under vacuum with a desiccating cryoprotectant and liquid nitrogen. Freeze-dried cells will be kept at -80°C until incorporated in the transport device, which will then remain at room temperature.

Transport device



A suitable transport device should facilitate safe transport of the ‘key’, be compact, portable and user-friendly and most importantly ensure cell viability.

Our design was inspired by the Mix2vial® system2 , which we are utilising to keep freeze-dried cells and LB medium separated in two containers, in a sterile environment. When the need comes to reactivate the bacteria, the two compartments can be easily connected, leading to the release of the LB medium. Our device streamlines the resuspension of a freeze-dried pellet of cells already transformed with our unique plasmids, driving cell growth and gene expression. The expression (fluorescence) profile of the key can then be measured using suitable detection equipment.

The device consists of the following parts:

• Top container: it is a hollow cylinder with a sealed top. It holds the LB medium and the bottom is sealed with an easily pierceable material, such as aluminium foil.
• Bottom container: it is a hollow cylinder with a sealed bottom. It holds the freeze-dried cells and the top is sealed with an easily pierceable material, such as aluminium foil.
• Middle piece: it works as a joint for the two containers and allows flow of medium from the top container to the bottom container. Inside this piece there are two cylinders, one pointing upwards and the other downwards, to pierce the aluminium seal once the containers are attached by screwing.
• Polystyrene Foam: designed to provide support to the parts inside the key set.
• Outer case: provides protection to the other parts.





Production



The production of the Key. coli transport device prototype is still being undertaken with the help of one of the 3D printing laboratory at the University of Nottingham.

In the finalised version of the Key. coli transport device, all parts except for the polystyrene foam, will be made of polypropylene. This will allow for sterilisation by autoclaving and, therefore, reuse.

Assembly and Activation



To assemble the device:

• Hold the middle piece with the top part up.
• Hold the medium container upside down and attach it to the middle piece, with two screwing movements.
• Hold the cell container with the seal facing upwards and attach it to the bottom of the middle piece with two screwing movements.
• Still in the same position, screw the cells container completely into the middle piece to destroy the seal.
• Screw the medium container completely to destroy the seal and allow liquid to flow into the cells container.
• Mix gently.
• Place the assembled device, with the cell container down, in a fixed surface (or incubator) until measurement.
• Once assembled, the device should be held with the top part up and should not be turned upside down.



Experimental planning



1. Develop freeze-drying and revival protocol for E. coli cells

2. Assess the effect of different storage temperatures on cell viability:

• Freeze-dried cells will be stored at room temperature (RT), 4oC and -80oC

3. Assess the effect of storage time on cell viability:

• Freeze-dried cells will be revived at different times after initial storage



4. Assess signal reproducibility in different conditions:



• Freeze-dried cells will be sent to other iGEM teams

1Morgan, C., Herman, N., White, P., & Vesey, G. (2006). Preservation of micro-organisms by drying; A review. Journal of Microbiological Methods, 66(2), 183-193. doi:10.1016/j.mimet.2006.02.017
2 Mix2Vial® Reconstitution System and Needle-Free Transfer Device. (n.d.). Retrieved November 01, 2017, from https://www.westpharma.com/products/reconstitution-and-transfer-systems/mix2vial-and-needle-free-transfer-device