Team:Freiburg/B subtilis
Our Model Organism
The human colon is home to a lot of tiny microorganisms. To be precise, our whole body contains 10 times more bacteria cells than our own endogenous cells1.
In most cases, these bacteria are not harmful to the human body. Although some organisms can cause infectious diseases, nearly 100% of our survey participant knew that this doesn’t mean bacteria are generally harmful.
The digestive system is host to several million of bacteria which are essential for digestion, proper absorbance of nutrients and the synthesis of vitamin K and B2.
The entire microbiota in the gut is a complex ecosystem that is not well understood yet3. Dis-balances in the composition of the intestinal flora can cause a variety of diseases.
We confronted different gastrologists with this topic and they confirmed that the flora is “essential” and “ really important since a disturbed intestinal flora would cause many diseases”.
But what exactly is disturbing this intestinal flora?
Studies have shown that changes in diets,operations and health condition will perturbate the microbiota and may lead to irritative bowel diseases, chronic inflammation, infectious diseases with harmful bacteria or malabsorption4.
Due to individual lifestyle and genetic dispositions, everyone has a distinct bacterial flora in his/her gastrointestinal tract.
To support and rebuilt the intestinal flora, people often grab for probiotics.
Asking the Ulcerative Colitis patients that filled out our survey, whether or not they have consumed probiotics before, almost half of the participant answered with yes. This result lets us assume that the concerns about taking our product too will not be insuperable.
Probiotics are nonpathogenic, living bacteria with health benefits for humans when consumed in a frequent manner5.
They play an important role in immunological, digestive and respiratory functions and even the number and type of probiotic in foods and drinks are increasing at the moment.6
Typical microorganism used as probiotics today are Lactobacillus, E.coli, Enterococcus and Bacillus subtilis7.
For example: the probiotic Biosporin contains Bacillus subtilis and inhibits the growth of Helicobacter pylori, a bacterium which is known to cause gastrointestinal diseases.8
In this year's iGEM project we, team Freiburg, decided to work with the model organism Bacillus subtilis because of its innocuousness, robustness and ability to build spores. We wanted to use this spores as a carrier for two tools in combination as our new form of targeted drug delivery system.
In our survey we have also asked people, if they think spores are generally harmful to the human body. In comparison to the same question about bacteria, the results are different. Nearly 18.8% answered with “Yes, spores are generally harmful”.
They play an important role in immunological, digestive and respiratory functions and even the number and type of probiotic in foods and drinks are increasing at the moment.6
Typical microorganism used as probiotics today are Lactobacillus, E.coli, Enterococcus and Bacillus subtilis7.
For example: the probiotic Biosporin contains Bacillus subtilis and inhibits the growth of Helicobacter pylori, a bacterium which is known to cause gastrointestinal diseases.8
In this year's iGEM project we, team Freiburg, decided to work with the model organism Bacillus subtilis because of its innocuousness, robustness and ability to build spores. We wanted to use this spores as a carrier for two tools in combination as our new form of targeted drug delivery system.
In our survey we have also asked people, if they think spores are generally harmful to the human body. In comparison to the same question about bacteria, the results are different. Nearly 18.8% answered with “Yes, spores are generally harmful”.
Bacillus subtilis has a rod shaped appearance and belongs to the family of the gram-positive bacteria.
Apart from being widely present in nature, it is also already a part of the microbial gut flora 9 and so a perfect candidate for being our probiotic. The US Food and Drug Administration (FDA) classifies Bacillus subtilis as a GRAS (Generally Regarded As Safe) organism 10. That means it is generally recognized as safe and can be used in S1 laboratories without problems.
Bacillus subtilis colonies have an irregular, large size with undulate margin. They have a white and dull colour and a dry texture.
Bacillus subtilis growth follows the typical 4 phases of bacterial growth (Figure 2). The start up phase is called lag phase which comprises the time the bacteria need to adapt to the new environment 11. With reaching the next phase, the exponential growth phase, the bacteria are dividing at their maximum division rate. Bacillus subtilis has a doubling time of 30 minutes under ideal conditions. This can be calculated with the slope of the exponential growth. In our experiment, the doubling time of the wild type is 28 minutes.
Figure 2 : Growth curve of the Bacillus subtilis strain WT168.
Figure 3: Exponential growth phase of Bacillus subtilis.
When all the substrate is consumed, the stationary stage begins where there is no more additional growth mensurable and the culture is at its highest density. Now the death-phase follows, where the bacterial culture starts to autolyse.
Bacillus subtilis is able to form endospores under distress, which is one of the most efficient adaptations to a lack of nutrients.
The sporulation begins with an asymmetrical cell division into the mother cell and the smaller prespore. The smaller prespore is now engulfed by the mother cell and the mother cell starts assembling the multiple layers of the spore. The cortex, a modified form of cell wall, is synthesized to give the spore their typical oval shape. At the same time the crust is formed, the spore coat begins to be deposit on the outside surface of the spore. The last part of the sporulation is called maturation, during this period the characteristics resistance, dormancy and germinability of the spores get established and the mother cell is lysed in order to release the endospore12. This spore is now highly resistant to heat, enzymatic attacks. UV-light and pressure and can re-enter its life cycle under the right conditions.
Furthermore it is a big advantage for our approach of working with surface fusion proteins, that the mother cell is able to synthesize the wanted proteins by itself and also assembles the proteins to the spore crust by itself. So the process of bringing a protein through a membrane is not needed. Through this, the cumbersome process of fusing proteins to the surface of artificial beads is obsolete. Since the outermost layer of the spore - the crust - is mostly build up of the proteins CotZ and CgeA 13, we use those to set up the fusion proteins. By fusing highly specific binding tools and enzymes to the crust proteins we found a new method of targeted drug delivery to reduce side effects occuring under systemic drug dispersal.
Furthermore it is a big advantage for our approach of working with surface fusion proteins, that the mother cell is able to synthesize the wanted proteins by itself and also assembles the proteins to the spore crust by itself. So the process of bringing a protein through a membrane is not needed. Through this, the cumbersome process of fusing proteins to the surface of artificial beads is obsolete. Since the outermost layer of the spore - the crust - is mostly build up of the proteins CotZ and CgeA 13, we use those to set up the fusion proteins. By fusing highly specific binding tools and enzymes to the crust proteins we found a new method of targeted drug delivery to reduce side effects occuring under systemic drug dispersal.
1. Guarner, F. , Malagelada, J. , Gut flora in health and disease , 6 Feburary 2003, p. 512
2. De Vrese, M. , Schrezenmeir, J. , Probiotics, Prebiotics, and Synbiotics, 07 May 2008, Volume 111 of the series Advances in Biochemical Engineering/Biotechnology pp 1-66
3. Benson, A.K. , Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors, 10 June 2010 18933–18938, doi: 10.1073/pnas.1007028107
4. Hayes, P. , Irritable Bowel Syndrome: The Role of Food in Pathogenesis and Management, 2014 Mar; 10(3): 164–174.
5. Guarner, F., Rerdigon, G., Should yoghurt cultures be considered probiotic?, 17 Januaey 2005, British Journal of Nutrition (2005), 93, 783–786
6. Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live LacticAcid Bacteria - Food and Agriculture Organization of the United Nations, 4 october 2001
7. Ouwehand, A. C. , Salminen, S. , Probiotics: an overview of beneficial effects, August 2002, Volume 82, Issue 1, pp 279–289
8. Duc, Le H., Characterization of Bacillus Probiotics Available for Human Use, 20 December 2003, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2004, p. 2161–2171
9. Qin, J. , Li, R. , A human gut microbial gene catalogue established by metagenomic sequencing, Nature 464, 59-65 (4 March 2010)
10. de Boer Sietske, A. & Diderichsen, B. Appl Microbiol Biotechnol (1991) 36: 1. doi:10.1007/BF00164689
11. Buchanan, R.L , Whiting , R.C, When is simple good enough: a comparison of the Gompertz, Baranyi, and three-phase linear models for fitting bacterial growth curves , doi:10.1006/fmic.1997.0125
12. Errington, J. , Bacillus subtilis Sporulation: Regulation of Gene Expression and Control of Morphogenesis , MICROBIOLOGICAL REVIEWS, Mar. 1993, p. 1-33
13. Driks, A., Bacillus subtilis Spore Coat , Microbiol. Mol. Biol. Rev. March 1999 vol. 63 no. 1 1-201 March 1999
