The spores of Bacillus subtilis will be contained in a compartment which is impermeable to bacteria and liquid, but can still let through volatiles. Metabolites, water, and alanine should be separated from the spores by a breakable membrane. Mixing these compounds will cause germination.
The material we use for our sticker is TPX®, or polymethylpentene. This polymer is available as thin, transparent sheets. The advantage is that it is relatively cheap, strong, and capable of letting through volatiles. The radius of the pores in TPX® is between 1 an 10 nm, which is at least 50x larger than the average badmeat-volatile, but still small enough to keep liquid and bacteria in (see the figure below).
Comparison of the size of the TPX pores, volatiles and Bacillus subtilis.
Design requirements
Material
The material should not break too fast and should resist a human grip strength with a minimum of 40 pounds pinch and pressure.
The product should be made of a material that is light and easy to handle.
The material should be durable and not too expensive.
The volatiles and oxygen should penetrate through the material, while the spores, bacteria and liquid should stay inside.
The material should fit in a meat package.
The material should not be toxic or become toxic for the bacteria
The material should be able to cope with a temperature of at least 125 Celsius.
Measurements
Needs a visible feedback system for the human eye and should easy to understand for the customer.
The product should keep the visible feedback stored over time.
Appearance
Product should be made of attractive colors and shape.
Safety
The bacteria should not escape from the sticker nor harm the environment. Therefore the product should provide adequate support and be reliable.
Customer comfort
Easy to use the visible feedback device.
The product may provide different degrees of strength depending on the development of the customer strength.
Development design
For the development of the sticker, the PDCA (Plan Do Check Act) strategy was used to develop an ultimate device that keeps track of the needs of the customer and the environment in the project design.
The following questions where raised:
Who is your external customer?
The customer who is against food spoilage and customers that are willing to buy it.
What value do you want to deliver to that customer?
A replacement for the "use by date and sell by date" system. We want to develop a new product that indicates when meat starts to spoil before the costumer can notice this by eye, by smell or by taste.
Who, in you iGEM group, delivers that value?
The design engineers and the biologists of iGEM Groningen 2012.
How do they deliver that value?
By doing research and creating a new indicator to predict when meat starts to get spoiled.
Material
Two different materials were carefully chosen for our final product. At first, the outer bag should be resistant enough to support the bacteria inside the sticker and give strength to the pressure that is applied by the customer. Its physical properties and characteristics should not be affected when too much pressure or deformation is applied on the sticker. It should be light and small for easy removing and placing in the meat package.
Second, the case should be made from a light material and the surface should be flat to provide grip and avoid sharp edges or other risks for the user.
Taking into account all these characteristics, we considered three materials for the sticker: polyetheen (sandwich bag), Polyvinyl chloride (cling film) and Polymethylpentene (PMP) als commonly known as TPX. We chose TPX as the material for the outer layer of the sticker, because this material fulfills all the requirements mentioned above.
In addition, we looked for another material needed for the inner compartment of the sticker, that separates the medium from the spores untill the customer wants to use it. At first, polyvinyl chloride turned out to be too weak for the outer layer of the sticker because it is easily broken. However, these properties are very suitable for the needed the breakable inner compartment of the sticker. Not a lot of pressure or strength is needed to break the inner compartment, which is handy for the customer when handling the sticker.
References
Krentsel B.A., Kissin Y.V., Kleiner V.I., Stotskaya S.S. Polymers and Copolymers of Higher a-Olefins, Hanser Publishers: New York, 1997.
H. C. Raine, J. Appl. Polym. Sci. 11, 39 (1969)
Mitsui Chemicals Co., Properties of Standard TPX Grades, 2004.
FDA CFR Title 21 Sec. 177.1520 Olefin polymers (C) 3.3b for TPX(4-methylpentene-1-based olefin copolymer).
Mathiowetz V, Kashman N, Volland G, Weber K, Dowe M, Rogers S (February 1985). "Grip and pinch strength: normative data for adults". Arch Phys Med Rehabil 66 (2): 69–74. PMID 3970660.
Tools and supply
Sealboy type: 236 SBSA-2, Serie: 0070133017 to affix the TPX material and cling film in the setting 7.
Sanyo labo autoclave MLS-3020U (RUG Serial: 4495) to sterilize the TPX material.
Gas profile 1 SCS, Part: 6.103.000, series: 0213372 to work sterile during fill-up the sticker.
B-D Plastipak 21G 11/2 40/8 NR2 2ml to fill up the sticker.
Cling film brand: Folia vershoudfolie, 50m width 29cm, 15 micrometer, oxygen permeability, fat- and waterproof. Inner compartment for the the sticker.
TPX X-44B#25 and X-44#50, Company: Mitsui Chemicals Co., Brand: TPX to use as outer layer from the sticker.
Luria Broth Brand: Lennox.
Prototype
The prototype of the inner compartment is made from cling film with a side affix of 1 mm thickness. The subsequent created inner compartment was filled with sterilized Luria Broth (LB). In the outer layer of the sticker was made of TPX filled with the cling film package and the spores, the 'seeds' of the Food Warden bacteria (link bacteria). An important feature that we had to take into account was the oxygen exchange between the TPX should and the LB growth medium. If this does not happen, the bacteria will not grow at its optimal rate. Therefore, we performed experiments by using closed flasks containing 50ml medium and 84ml air to calculate the minimum oxygen exchange. In this experiment the bacterium growth in 37 degree Celsius. The 50 ml medium in the flasks has a diameter of average 6cm where 84ml of air average of 21% oxygen at a starting point is applied on the medium in the flask. That makes 17.64ml of total oxygen that is needed for the growth of 50ml medium. From the TPX characteristics and mathematical calculations we made the following graph picture 2.
Picture 2: This graph represent the minimum surface cm2 is needed per ml. The green line is TPX 50 um thickness and the red line is 25um thickness. The x-axis is the volume of ml Luria broth with the Food warden bacterium. The y-axis is the surface in cubic cm at 37 Celsius degree what is needed per ml.
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