Difference between revisions of "Team:Freiburg/B subtilis"
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| − | The digestive system is host to several millions of bacteria which are essential for digestion, proper absorbance of nutrients and the synthesis of vitamin K and | + | The digestive system is host to several millions of bacteria which are essential for digestion, proper absorbance of nutrients and the synthesis of vitamin K and B<sup>2</sup>. |
| − | The entire microbiota in the gut is a complex ecosystem that is not well understood yet | + | The entire microbiota in the gut is a complex ecosystem that is not well understood yet<sup>3</sup>. 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”. (Quote: ask the gastrologists again if we are allowed touse their names ) | 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”. (Quote: ask the gastrologists again if we are allowed touse their names ) | ||
But what exactly is disturbing this intestinal flora? | 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 malabsorption | + | 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 malabsorption<sup>4</sup>. |
Due to individual lifestyle and genetic dispositions, everyone has a distinct bacterial flora in his/her gastrointestinal tract. | 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. | To support and rebuilt the intestinal flora, people often grab for probiotics. | ||
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| − | Probiotics are nonpathogenic, living bacteria with health benefits for humans when consumed in a frequent | + | Probiotics are nonpathogenic, living bacteria with health benefits for humans when consumed in a frequent manner<sup>5</sup>. |
| − | Typical microorganism used as probiotics today are Lactobacillus, E.coli, Enterococcus and Bacillus subtilis | + | Typical microorganism used as probiotics today are Lactobacillus, E.coli, Enterococcus and Bacillus subtilis<sup>6</sup>. |
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. | 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. | ||
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Bacillus subtilis has a rod shaped appearance and belongs to the family of the gram-positive bacteria. | 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 7 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 8. That means it is generally recognized as safe and can be used in S1 laboratories without problems. | + | Apart from being widely present in nature, it is also already a part of the microbial gut flora <sup>7</sup> 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 <sup>8</sup>. 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 colonies have an irregular, large size with undulate margin. They have a white and dull colour and a dry texture. | ||
[Bild davon] | [Bild davon] | ||
| − | 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 9. 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. | + | 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 <sup>9</sup>. 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. |
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| − | 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 | + | 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 endospore<sup>10</sup>. 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. | 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 11, we use those to set up the fusion proteins. | + | Since the outermost layer of the spore - the crust - is mostly build up of the proteins CotZ and CgeA <sup>11</sup>, 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. | 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. | ||
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References:<br> | References:<br> | ||
Revision as of 18:35, 16 October 2016
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 cells 1.
In most cases, 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 millions 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”. (Quote: ask the gastrologists again if we are allowed touse their names )
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.
Typical microorganism used as probiotics today are Lactobacillus, E.coli, Enterococcus and Bacillus subtilis6.
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 7 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 8. 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.
[Bild davon]
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 9. 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.
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 endospore10. 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 11, 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.
When we started to work with this versatile model organism, we noticed that in our surrounding no one is working with Bacillus subtilis. That’s why we had to reach wider and are glad to got help from Julia Bartels, a former member of the iGEM Team Munich 2012. To ease the work for future iGEM Teams we started a Bacillus subtilis manual together with iGEM Bonn 2016.
Here are 12 AMAZING Facts about Bacillus subtilis we hope you enjoy:
1. Bacillus subtilis is one of the few bacteria strains which can build endospores.
2. B. subtilis lives all over the world, mostly in soil.
3. Its spores were sent to outer space for one and a half years and were still able to germinate back on earth.
4. B. subtilis creates strong foot odor.
5. If you’d grow a single B. subtilis cell for 100 generations you’d receive 1030 cells after around a day and would need 1018 liters of media. (luckily they starve with a density of circa one billion cells per milliliter, so don’t panic)
6. B. subtilis is used for synthesis of antibiotics. ( such as Subtilin)
7. The core of its endospores is very dry and resistant to moisture.
8. B. subtilis is an ingredient of washing powder. (can produce proteases & amylases)
9. B. subtilis is used as a natural herbicide due to its ability to produce lethal substances.
10. B. subtilis is widely used as a probiotic. (supports your health especially the digestive system)
11. Once a group was able to resume some spores they found in a 250 million years old salt crystal and were able to “revive” them. (not B. subtilis spores though)
12. Spores are truly amazing!
1. Bacillus subtilis is one of the few bacteria strains which can build endospores.
2. B. subtilis lives all over the world, mostly in soil.
3. Its spores were sent to outer space for one and a half years and were still able to germinate back on earth.
4. B. subtilis creates strong foot odor.
5. If you’d grow a single B. subtilis cell for 100 generations you’d receive 1030 cells after around a day and would need 1018 liters of media. (luckily they starve with a density of circa one billion cells per milliliter, so don’t panic)
6. B. subtilis is used for synthesis of antibiotics. ( such as Subtilin)
7. The core of its endospores is very dry and resistant to moisture.
8. B. subtilis is an ingredient of washing powder. (can produce proteases & amylases)
9. B. subtilis is used as a natural herbicide due to its ability to produce lethal substances.
10. B. subtilis is widely used as a probiotic. (supports your health especially the digestive system)
11. Once a group was able to resume some spores they found in a 250 million years old salt crystal and were able to “revive” them. (not B. subtilis spores though)
12. Spores are truly amazing!
References:
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. Ouwehand, A. C. , Salminen, S. , Probiotics: an overview of beneficial effects, August 2002, Volume 82, Issue 1, pp 279–289
7. Qin, J. , Li, R. , A human gut microbial gene catalogue established by metagenomic sequencing, Nature 464, 59-65 (4 March 2010)
8. de Boer Sietske, A. & Diderichsen, B. Appl Microbiol Biotechnol (1991) 36: 1. doi:10.1007/BF00164689
9. 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
10. Errington, J. , Bacillus subtilis Sporulation: Regulation of Gene Expression and Control of Morphogenesis , MICROBIOLOGICAL REVIEWS, Mar. 1993, p. 1-33
11. Driks, A., Bacillus subtilis Spore Coat , Microbiol. Mol. Biol. Rev. March 1999 vol. 63 no. 1 1-201 March 1999
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. Ouwehand, A. C. , Salminen, S. , Probiotics: an overview of beneficial effects, August 2002, Volume 82, Issue 1, pp 279–289
7. Qin, J. , Li, R. , A human gut microbial gene catalogue established by metagenomic sequencing, Nature 464, 59-65 (4 March 2010)
8. de Boer Sietske, A. & Diderichsen, B. Appl Microbiol Biotechnol (1991) 36: 1. doi:10.1007/BF00164689
9. 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
10. Errington, J. , Bacillus subtilis Sporulation: Regulation of Gene Expression and Control of Morphogenesis , MICROBIOLOGICAL REVIEWS, Mar. 1993, p. 1-33
11. Driks, A., Bacillus subtilis Spore Coat , Microbiol. Mol. Biol. Rev. March 1999 vol. 63 no. 1 1-201 March 1999
