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From 2012.igem.org

Our construct idea is simple and effective: there will be a production of pigment under the regulation of rotten-meat reactive promoter. When the Bacillus subtilis senses the volatiles from the rotten meat, the rotten meat promoter becomes active thus allowing the production of downstream genes. We put pigment genes available downstream of the promoter so that the pigment would be produced when the promoter is activated.

We use our Bacillus subtilis backbone (BBa_K818000) that has sacA and chloramphenicol resistance gene for chromosomal integration and transformants antibiotic screening. This backbone also has E. coli origin of replication, so it can be amplified inside E. coli.

There are several ideas to improve our current construct, such as: multi-colored pigment system and fine tuning of the pigment production.


Tuning system of Pigment Production

The core concept behind the Food Warden is that it should pave the way to a more comprehensive, scientifically informed prediction of food edibility that goes beyond conventional best-before dates. The Food Warden as it is now is only a proof of principle. The goal is then to produce a system that is truly more accurate and reliable than the best-before date. The tuning needed requires a comprehensive study on the relationship between volatile concentration, degree of spoilage health risk and pigment production:

1. Volatile concentration: Building upon our gas chromatography approach in order to quantitatively assess the volatile production of spoiling meat.
2. Degree of spoilage heath risk: The unrefined nature of current assessments of spoiling degrees in food (see https://2012.igem.org/Team:Groningen/Stop_the_food_waste_initiative) makes this a difficult step. A time resolved total microbial count analysis could be done to assess edibility in terms of that standard for specific types of meat.
3. Pigment production: The control of pigment production dynamics will depend on the outcomes of the previous two aspects of the tuning procedure. Once the relationship between volatile composition/concentration and health risk is elucidated to some degree, the pigment production can be tuned to fit this parameter.

The pigment production can be tuned to a desired speed and sensitivity with different regulating promoter, different rbs, and with positive feedback system to increase the pigment production. One example of the positive feedback system that can be applied to increase pigment production under the regulation of the rotten meat promoter:

When the rotten meat promoter is activated, the pigment and inducer will be produced. The positive feedback loop is then formed so that the pigment and the inducer will be in the loop, increasing the production rate of the pigment. This system is meant to increase the production speed of the pigment. One of the possible set of inducible promoter-inducer is pRE promoter (BBa_K116603) with CII (BBa_K116602).


Multi-colored Pigment System

The pigment system consists of two signals, which are quite simply 'on' or 'off'. There are some disadvantages to this in terms of user-friendliness that need to be addressed. The Food Warden can only do its job if it can grow properly upon breaking of the inner compartment of the sticker. It is plausible that manufacturing errors during the production of an eventual Food Warden product could lead to issues with the germination of the spores, resulting in a sticker that does not do its job. Eventualities include:

1. Incorrect medium: In the event that the medium supplied in the sticker is not of the correct composition, the Food Warden will not grow, and therefore will not be able to identify spoiling meat.
2. Absence of viable spores: A defect sticker could be accidentally produced that either lacks spores capable of germination or lacks spores entirely.
3. Contaminant organism: It is conceivable that the spores could be outcompeted in a medium that If the user is not aware of these problems he or she may assume that the lack of pigment production simply means the meat in question is not spoiling yet, whereas it may already be spoiling and indeed will without any warning.

Although these eventualities are a production process concern, it is our job to produce a system that minimizes the risk of these problems affecting the user. Therefore, we would have liked to build in a positive control that allows the user to confirm that the Food Warden in their sticker germinates and grows. The positive control considered was the inclusion of a constitutively produced second pigment. This pigment serves as a signal to the user that the Food Warden is functional upon germination. This positive control pigment should of course not interfere with the visual detection of the warning pigment. To ensure this, the following option were considered:

1. Choosing the two pigment colors such that the warning pigment color is highly dominant over the control pigment color.
2. Placing the pigment under the control of a weak constitutive promoter, producing the control pigment at minimum levels required for user detection, therefore more easily being overpowered by the warning pigment.
3. Placing the pigment under the control of a constitutive promoter, identified in our microarray analysis, that down-regulates the gene it controls under spoiling meat conditions, allowing the warning pigment to more easily overpower the control pigment. [[[diagram listing a few down regulated promoters we identified that we could use]]]
4. Including an operator for the transcription of the control pigment that allows for repression by a repressor protein. This repressor protein would be under the control of the same promoter responsible for warning pigment transcription.
The construct can be put as following:




5. Placing a control pigment that reacts to specific compound to change its color. The control pigment is under the regulation of a constitutive promoter while the rotten meat promoter regulates the compound needed to change the control pigment's color.
The construct can be put as following:




When the rotten meat is activated, the compound needed to change the control pigment's color and the repressor will be produced. The control pigment production will be stopped and the existing control pigment will start to react to the compound, changing its color and becoming the warning pigment. This can be achieved using existing biobrick: BBa_K274100 (lycopene, red pigment) and adding the compound (CrtY) encoded by BBa_K118008 to make the red color from lycopene into yellow color (beta-carotene). Another construct using positive feedback loop for the warning-pigment-compound without stopping the control pigment production is as following:




In this system, the control pigment will still be produced even when the rotten meat promoter activates. The production speed of the second pigment compound which reacts to the control pigment to create new color is enhanced by the positive feedback loop, so the warning pigment color can be immediately produced.

This idea basically mimics the traffic light function: different colors production for every state of the meat. When the meat is still fresh, a specific pigment will be produced. When the meat starts to rot, another pigment will be produced overriding the previous pigment.