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Latest revision as of 03:50, 27 October 2012

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 coming off the spoiling meat 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 what we came up with:

Bacillus subtilis would be an ideal candidate as a 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. This restorative process is germination. For the information, take a look here and here. Because of this ability to go dormant, our bacterium can be stored and activated whenever it is needed!

However, this does not solve the problem of the bacteria/spores next to the food. That's why we designed 'the sticker', a containment device to store and activate the spores at the right time. For clarity: our team keeps calling it 'the sticker' all the time. The sticker consists of two nested compartments. 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 come 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 nm and 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.

1. Siebring J. 2012 (unpublished)

Comparison of the size of the TPX® pores, volatiles and Bacillus subtilis.

Design requirements

Material

The material should not break easily 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 inexpensive.
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 that should easy to understand for the consumer.
The visible feedback should not degrade 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 consumer 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 people that buy meat on regular basis.

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 spoil.

Plan

Our first plan is to identify exactly what we have to do to make a sticker. The sticker should be the same as the use of a glow-in-the-dark stick, that you first have to break before it gives off a bright chemical light. Everyone should understand how it works. The indication color might be made with the same colors as a traffic light, to indicate when the meat is fresh, a bit spoiled or really spoiled. During our second case, we want to produce an easily activated sticker. In the third part of the plan, the outer sticker should be strong enough, let volatiles go through the outer layer of the sticker, and not any bacteria or liquid should go out of the outer layer of the sticker.

Do

The following solution was chosen: Build a transparent plastic bag with a smaller compartment inside, where the medium inside is visible for the user when the color changes. Make the inner compartment of a weaker material where the sides break when a pinch force is applied.

The following pilots where made:

1. Make sticker user-friendly concerning the breakage of the inner compartment.
2. Test if the bacteria grow in the sticker.
3. Storage and activation in the sticker.
4. Test if the medium with the Food Warden bacteria will produce pigment in the sticker.
5. We made a set-up with pumps to check the influence of oxygen poor and oxygen rich environments on the sticker.
6. Oxygen concentration tested in jars with rotten meat at different temperatures.


Check

Depending on the success of the pilots, the number of areas for improvements and the scope of the whole initiative, we decided 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, you can move on to the final phase.

Act

After implementing 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) also 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 consumer 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 breakable inner compartment of the sticker. Very little pressure is needed to break the inner compartment, so it will be easy to start the Food Warden system when the consumer needs it.

1. Krentsel B.A., Kissin Y.V., Kleiner V.I., Stotskaya S.S. Polymers and Copolymers of Higher a-Olefins, Hanser Publishers: New York, 1997.
2. H. C. Raine, J. Appl. Polym. Sci. 11, 39 (1969)
3. Mitsui Chemicals Co., Properties of Standard TPX Grades, 2004.
4. FDA CFR Title 21 Sec. 177.1520 Olefin polymers (C) 3.3b for TPX(4-methylpentene-1-based olefin copolymer).
5. 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 supplies

1. Sealboy type: 236 SBSA-2, Serie: 0070133017 to affix the TPX material and cling film in the setting 7.
2. Sanyo labo autoclave MLS-3020U (RUG Serial: 4495) to sterilize the TPX material.
3. Gas profile 1 SCS, Part: 6.103.000, series: 0213372 to work sterile during fill-up the sticker.
4. B-D Plastipak 21G 1^1/2 40/8 NR2 2ml to fill up the sticker.
5. Cling film brand: Folia vershoudfolie, 50m width 29cm, 15 micrometer, oxygen permeability, fat- and waterproof. Inner compartment for the the sticker.
6. TPX X-44B#25 and X-44#50, Company: Mitsui Chemicals Co., Brand: TPX to use as outer layer from the sticker.
7. 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 bacterial spores. An important feature that we had to take into account was the oxygen exchange between the TPX® and the LB growth medium. If this does not occur extensively, the bacteria will not grow at their optimal rate. Therefore, we did 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 degrees 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.

This graph represents 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 containing the Food Warden bacterium. The y-axis is the required surface area (cubic cm) per mL at 37 degrees Celsius.

Test

Make sticker user-friendly concerning the breakage of the inner compartment.

We want to check if each different volume is equally easy to break.

Make sticker user-friendly concerning the breakage of the inner compartment.

Test if the bacteria grow in the sticker.

All different volumes just after breakage, the LB broth has no traces of bacterial growth.

Bacteria grow inside the sticker.

We tested also different kinds of volumes to verify the calculations in the surface area plot.

Storage and activation of Bacillus subtilis spores

Spores placed in the sticker.

One of bases of our project is the idea that spores can be stored inside the sticker and activated when needed. However, germination only occurs when conditions are favorable for the bacteria to grow. To test whether this condition can be made inside the sticker a very simple experiment was done: germination of spores inside a sticker. Several stickers were made that contained B. subtilis spores in one compartment and normal luria broth medium in the second compartment. The sticker was stored for 1 day to prove that it is able to store them. On the second day, the compartment with medium was broken, thus mixing the medium with the spores. After a day on room temperature, the growth of B. subtilis inside the stickers was observed. This proves that the spores can be stored inside the sticker and that they can be activated at will.

Test if the medium with the Food warden bacteria will produce pigment in the sticker.

No growth in the sticker when it was placed in a bottle with rotten meat first (probably due to oxygen depletion), therefore we placed the sticker in a bottle with fresh meat

Extra - Test if the medium with the Food warden bacteria will produce a pigment in the sticker.

Bottle of rotten meat connected to the microarray set up to ensure the oxygen is pumped around

Test if the medium with the Food Warden bacteria will produce a pigment in the sticker.

First picture of the Bacillus producing the purple AmilCP pigment in the sticker and not in a bottle of the microarray set up.

Extra - Test if the medium with the Food Warden bacteria will produce a pigment in the sticker.

Sticker oxygen input test setup
To test the stickers with different amounts of oxygen supplied, a system was built with two pumps. One pump was used for the regular flow of meat volatiles. The other pump was used for pumping fresh air into the flask with the stickers. These pumps alternate in activity so that every 15 minutes fresh air is supplied for 3 minutes. Two different systems were made, one where the air input is applied directly into a jar with stickers and one where the jar with stickers is placed at a different spot so it obtains some fresh air but far less. The setup is shown below:

Left: pump 1 is turned on. The air flows through the system as is indicated by the arrows. Both flasks with stickers are provided with the same amount of volatiles from the meat. Pump 2 (turned off) is connected to the fresh air. Right: Pump 2 is turned on. Fresh air flows in the flask with stickers as is indicated with the arrows. The other flask only receives a small amount of fresh air when pump 1 is turned back on and the remaining air is pumped through the whole system.

We also used stickers with different plastic thickness. The tested stickers were 25µm and 50µm. The results show a clear result that the stickers supplied with plenty of fresh air and a thickness of 25µm already produce color in 1 day. The stickers with the thicker plastic produced color one day later. For the low fresh air supply color was produced after 3 days with the 25µm plastic. The 50µm plastic took one day longer to produce color.

The first series of sticker experiments let us believe that oxygen limitation might play a role in the growth of B.subtilis inside the sticker. We devised an experimental setup that would allow us to monitor the oxygen level inside a closed system with rotten meat. Doing triplo tests, in which a 100 ml bottle filled with about 30 grams of meat is placed at 20 or 37°C. By taking a 1ml air sample from the bottle each hour the oxygen level of this closed system is measured for both conditions over time. Each hour the air sample was injected in a closed chamber with a Clark electrode. This gas-phase oxygen electrode system makes a measurement of the oxygen level. The oxygen sample was analyzed with a certificated ISO 6141 from the Linde group, using 21% and 2% oxygen as references and N2 as a base line reference. The data was taken over 24 hours and plotted in a graph. From this graph it was noticed that the oxygen levels drop from 21% to ≈11% in a 20°C environment and to ≈3% in a 37°C environment. Because our initial sticker design was based on a 21% oxygen environment, a change of the surface size of the sticker is needed to ensure enough oxygen exchange inside the sticker. In the second series of sticker tests, we already made the surface larger to prevent this limitation from giving any problems during the testing. Using this data we can design stickers that can compensate for lower oxygen levels, thus ensuring that there is no limitation on germination, growth and pigment production.

Oxygen percentage graph from meat in 20°C and 37°C.

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