Not everyone likes free living, engineered bacteria next to their food. But in order to make our Food Warden system work properly, our bacteria should be able to react to the volatiles of meat that starts to spoil while it is located in, for instance, a meat package. We had a lot of discussions about how to achieve a very safe and easy-in-use solution, and this is where we came up with:
Bacillus subtilis would be a ideal candidate as chassis for our genetically engineered construct, because it has the ability to form endospores, a kind of dormant state that they use for survival. They can survive high levels of heat (>100 °C), drying, radiation, and many damaging chemicals and simply be brought back 'to life' under the influence of sufficient nutrients and water, which is called germination. For the information, take a look here. Because of the germination, our bacterium can be stored and activated when we need it to be!
However, this does not solve the problem of the bacteria/spores next to the food. That's why we designed 'the sticker', a containment to store and activate the spores at the right time. And for clarity: our team keeps calling it 'the sticker' all the time. The sticker consists of two compartments that enclose eachother. The inner compartment contains a calibrated amount of nutrients, while the outer semi-permeable capsule contains the spores of our engineered strain. Breaking the barrier between the two compartments allows germination and growth of Bacillus subtilis cells.
Now the properties of the material we use for our sticker comes into play. The polymer we used is TPX®, also called 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 or spores in (see the figure below). More detailed information about the sticker design and its experiments, are stated 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 degrees Celsius.
Measurements
We want 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 have attractive color(s) and recognizable shape.
Safety
The bacteria should not escape from the sticker nor harm the environment or the costumer. 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.
Drawing of PDCA circle.
Plan
First, identify exactly what we have to build and to make a sticker. The sticker should be the same as the use of a light stick what you first have to break and than give chemical light color, that is visible and each dummy understand it how it works. The indication can be the same as a traffic light to indicate when meat is spoiled. The second case is that not allot of force should be applied by human pinch to get the sticker to break and work. The third part the the outer sticker should be strong and volatiles should go throw in the outer layer of the sticker and any bacteria or liquid should go out of the outer layer of the sticker.
Do
The following solution was choice n:
Build a transparent plastic bag with inside a smaller compartment, where the medium inside is visible for the user when the colour changes.
Make a sticker inner compartment of a weaker material where the side affix break when a pinch force is applied.
Concept drawings of the sticker.
The following pilots where made:
1. Make sticker user friendly to break the inner compartment.
2. Test if bacteria crow in the sticker.
3. Test the sticker if the medium with the Food warden bacteria make a pigment in the sticker.
Check
Depending on the success of the pilots, the number of areas for improvement you have identified, and the scope of the whole initiative, we made decide to repeat the "Do" and "Check" phases, incorporating our additional improvements.
Once you are finally satisfied that the costs would outweigh the benefits of repeating the Do-Check sub-cycle any more, you can move on to the final phase.
act
After implement our solution we generated part of a continuous improvement initiative, we made a loop back to the Plan Phase, and seek out further areas for improvement.
Material
Two different materials were carefully chosen for our final product. At first, the outer compartment should be resistant enough to support the bacteria inside the sticker and give resistance 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, to ensure the capsule is easily placed in meat package, for instance.
Second, the case should be made from a light material and the surface should be flat to provide grip and avoid sharp edges or any 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 most suitable material for the outer layer of the sticker, because this material fulfills all the requirements mentioned above.
In addition, we searched for another material needed for the inner compartment of the sticker, that separates the growth 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 breakable inner compartment of the sticker. Not a lot of pressure or strength is needed to break the inner compartment, so it will be easy to start the Food Warden system when the customer needs it.
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, made of TPX, was 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 occur extensively, the bacteria will not grow at its optimal rate. Therefore, we performed several experiments to calculate the minimum oxygen exchange. We used closed flasks containing 50ml medium and 84ml air to test the bacterial growth at 37 degree Celsius. The 50 ml medium in the flasks had a diameter with an average of 6 cm 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.
Test
Make sticker user friendly to break the inner compartment.
Test if bacteria crow in the sticker.
Test the sticker if the medium with the Food warden bacteria make a pigment in the sticker.
Results
Make sticker user friendly to break the inner compartment.
Movie: In the movie we see how easy the sticker to breaks.
Test if bacteria crow in the sticker.
We tested different kind of volumes and we achieve to make them.
Test the sticker if the medium with the Food warden bacteria make a pigment in the sticker.
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