Team:Groningen/Biofilm

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==Biology==
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==Biofilm==
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In nature, bacteria occur predominantly in highly organized multicellular communities called biofilms. Biofilm formation induces a complex developmental process, where cells differ from each other spatially and morphologically. The bacterial cells in such biofilms are composed of phenotypically different bacteria, demonstrating an intriguing example of heterogeneous regulation within an isogenic culture. Gram-positive bacteria have developed different strategies for survival in unfavorable environments, e.g. by getting competent or by sporulating. Biofilms offer an opportunity for the cells to survive extreme conditions as the cells in biofilms are more resistant to antibiotics and harsh circumstances.
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===Summary===
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[[Image:structure.jpg]]
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In our project we want our host bacterium to not only produce the coating material, but also apply it. Therefore we chose ''Bacillus subtilis'' as our host bacterium. ''B. subtilis'' can form a rigid biofilm that will cover the target surface before producing the [http://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [http://2010.igem.org/Team:Groningen#/biofilm_model model] on the biofilmformation, but furthermore we looked into ways to easily apply ''B. subtilis'' to the surface and let it form a biofilm there. One way to do this is by adding corn starch to regular TY-medium, making it an easily applicable paste.
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A rok bio film showing a high degree of structural complexity
 
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Bacillus subtulis a an ideal cantidate for a bio film coating since it is fast growing and has a very rigid extracellular matrix.
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===Introduction===
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<html>
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<div style="text-align: justify">
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</html>
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[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]
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Using biobased materials in the application or manufacturing of coatings has been the topic of many researches. However, using bacteria to make a coating substance and, most importantly, letting it do the coating process for you is something new. In our hydrophobofilm project we aim to use the extracellular fibrous proteins, DNA and polysaccharides that are formed in a biofilm, as a host matrix to embed our coating material, which in our case are hydrophobic proteins.  
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TasA
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Growing a biofilm on a surface as a way of coating it, might seem like a bad idea, since there are quite a lot of coatings out there to prevent biofilms forming in the first place. But why not "fight fire with fire”, and create a biofilm that is non-pathogenic and prevents other biofouling from taking place.
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Hydrophobins
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==Biology==
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''Bacillus subtilis'' is an ideal candidate for a biofilm coating. Firstly because it is quickly grows a biofilm which has a smooth extracellular matrix. Secondly, the bacterium is a well known and extensively studied model organism which makes is easier to work with. Finally ''B. subtilis'' is a gram-positive bacterium like ''Streptomyces coelicolor'', the bacterium that naturally produces hydrophobins. This might be an advantage when expressing and assembling the chaplin proteins in our host.
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The hydrophobic proteins that we are expressing in our bacillus biofilm are called chaplins and are derived from streptomyces. We streptomycyes is sporulating hyfe are trying to grow into the are. Since these hyfe are very small it is very hard to break the water air barrier for these hyfe, this is why streptomyces produces small hydrophobic proteins on the surface of it hyfe.
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<br>
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===Biology===
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<br>
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''In nature, bacteria occur predominantly in highly organized multicellular communities called biofilms. Biofilm formation involves a complex developmental process, where cells differ from each other spatially and morphologically. The bacterial cells in biofilms are phenotypically different, demonstrating an intriguing example of heterogeneous regulation within an isogenic culture. Gram-positive bacteria have developed different strategies for survival in unfavorable environments, e.g. by getting competent or by sporulating. Biofilms offer an opportunity for the cells to survive extreme conditions as the cells in biofilms are more resistant to antibiotics and other harsh circumstances like physical stress, drought or competing organisms. ''Bacillus'' even forms highly complex biofilms with a large degree of structural complexity and diversification of cell function within the biofilm. There are even channels within the biofilm to allow drainage of waste and diffusion of oxygen deep within the biofilm.(''Akos Kovacs)
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'''Three subgroups'''
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<br>
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In total their are eight different chaplins. These eight chaplins can be devided in to three groups.
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Chaplin A-C
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===Biofilm formation===[[Image:strain rok.jpg|right|500px]]
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This group of chaplin are the largest and are almost three times these size of the other chaplins ones around ... kD. What makes these chaplins special besides their size is that they have a cell wall anchor and a hydrophilic region as well as a hydrophobic region.  
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Biofilm formation usually starts with the accumulation of biomass, next there is the adhesion to a surface by the production of adhesion proteins. Then the production of "extracellular polymeric substances" (EPS) starts and the phenotypic diversification. After maturation of the biofilm sporulation kicks in. Since the pathways involved in biofilm formation in ''B. subtilis'' are just starting to be unravelled, not everything is known about the complex physiological interactions within a biofilm. By using an already existing pathway in ''B. subtilis'' for the auto-induction of our hydrophobic proteins, we try to minimize the amount of tinkering to the existing signaling pathways. Thereby leaving the natural system intact.  
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Chaplin D, F-H
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Timing is one the most important factors in successful assembly of our chaplins in EPS.
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These chaplin are small and have only a hydrophobic region
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''B. subtilis'' produces a protein that forms amyloidfibers called TasA. TasA is a very important protein to provide structural integrity in ''B. subtilis'' biofilms and is formed in the late stage of biofilm formation. The amyloid fibers that are formed provide the biofilm with an increased degree of rigidity (Romero et al, 2009). [http://2010.igem.org/Team:Groningen#/hydrophobins Chaplins] also assemble into amyloid fibers and provide a similar function in the hyphae of ''S. coelicolor'' (Cleassen et al, 2009), giving the hyphae the structural ability to grow high up in the air. Incorperating the chaplins at the same moment as TasA is formed would maximize the chance of successful assembly of chaplins in the EPS, while enabling maximum biofilm coverage. For more details on our expression pathway check out our [http://2010.igem.org/Team:Groningen#/expression expression] or [http://2010.igem.org/Team:Groningen#/modeling modeling] page.
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Chaplin E
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<br>
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This chaplin is also small but is in the vivo relaesed outside the cell to start the watertension lowering before the hyfe actually penetrates the water-air barrier
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[[Image:agar TY corn starch.jpg|right|300px]]
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====Coating surfaces===
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'''Physical properties'''
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Prevention from our biofilm to grow out of control, is an important aspect when you would apply the hydrophobofilm outside the lab. To deal with these
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What makes these proteins inteesting is that they are amfipathic, meening that they "in theory" can change hydrophilic surfaces into hydrophobic surfaces but in turn can also change hydrophobic surfaces into hydrophilic surfaces.  
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[http://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [http://2010.igem.org/Team:Groningen#/killswitch_model kill switch] for our hydrophobofilm. This kill switch relies on the production of a toxin and anti toxin. Where the anti toxin has a slightly shorter half-life than the toxin, thereby eventually resulting in the toxification of the cell itself. This toxification would occur after maturation of the biofilm. After the autotoxification the cells, the EPS with the embedded chaplin proteins will dry out, leaving a hydrophobic EPS layer on the surface.
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Chaplin are functional amyloids that will asemble by a catalytic process from monomers in polymerics chain forming rod like structure surfaces called amyloid fibers. These fiber are very rigid and and hard to break down. These fiber can only be broken up by boiling them in SDS.
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share distinguishing features with the medically important pathogenic amyloid fibers that are the hallmark of many neurodegenerative diseases such as Alzheimer's, Huntington's, systemic amyloidosis and the prion diseases.  
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<br>
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'''Biofilms as a biological coating'''
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[[Image:biofilm on ceramics.jpg|left|200px]]
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Applying our bacteria effectively to a surface poses big challenges.  such as, how to coat a surface in a short period of time, with low cost and low tech methods. Furthermore there must be enough nutrients for the organisms to successfully form a biofilm, yet you do not want to smear you surface in to much medium, so to avoid that the organism will only adhere to the medium and not to the surface itself. 
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The idea to make a biological coating has quite some advantages. For once biological coatings are quick to grow and the raw materials necessary for bio film growth are low in cost and plentiful available. So far there have been many coatings with biological substances but never before has bacteria been used for not only producing the coating material but also executing the coating process thereby saving a lot of effort.
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<br>
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For our bio film we have chosen bacillus subtiulus
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[[Image:biofilm ceramics total.jpg|right|ceramics]]
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'''Antifouling coating'''
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''Biofilm paste''
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We attempted to make a medium that could be easily applied to a surface and enable biofilm formation to take place. To achieve this we tried to make our medium more viscous. By adding corn starch to regular TY medium we increased the viscosity of our medium and also made it richer in nutrients. We [http://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations.
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We have created an easily applicable paste, to grow our biofilmcoating on all kinds of different surfaces. Another effect of the addition of cornstarch to the medium is an increased growing speed.
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Envirmentally friendly antifouling coating is
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<br>
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When marine micro-organisms like algea or poks adhere to the hall of ships they form a layer greatly increasing drag in the water. This results in higher fuel costs and increased erosion. To prevent organism to form to the hall of ships chemicals often containing copper and tin, are used in antifouling paints. A lot of these chemicals eventually end up in the oceans ecosystems accumulating in all trophic levels of marine life and contaminating estuarial silt near shipping routes . Estimates show that in the Netherlands alone, approximately 19 tuns of organotin and 30 tuns of copper end up in the environment every year.
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''A & B: B. subtilis biofilms grown overnight on ceramics coated with the biofilm paste. C: B subtilis biofilms dried out over four days, after formation.''
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Because of these environmental objections to chemical antifouling coatingds our hydrophobic bioflm would be a great ecological alternativ. This hydrophobic bio film will prevent the adhesing of spores and plankton to ship halls. Thereby preventing the growth of marine fouling organisms.
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<br>
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Waterresistance
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==References==
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<small>
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1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms
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Antfungal
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2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus
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subtilis Biolms
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Medical coatings
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3. Dennis Claessen, Rick Rink, Wouter de Jong, et al, 2009,  A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils
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Dispersants
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</small>

Latest revision as of 01:41, 17 November 2010

Biofilm

Summary

In our project we want our host bacterium to not only produce the coating material, but also apply it. Therefore we chose Bacillus subtilis as our host bacterium. B. subtilis can form a rigid biofilm that will cover the target surface before producing the hydrophobic proteins. As part of our project we made a model on the biofilmformation, but furthermore we looked into ways to easily apply B. subtilis to the surface and let it form a biofilm there. One way to do this is by adding corn starch to regular TY-medium, making it an easily applicable paste.


Introduction

B. sub Rok biofilm

Using biobased materials in the application or manufacturing of coatings has been the topic of many researches. However, using bacteria to make a coating substance and, most importantly, letting it do the coating process for you is something new. In our hydrophobofilm project we aim to use the extracellular fibrous proteins, DNA and polysaccharides that are formed in a biofilm, as a host matrix to embed our coating material, which in our case are hydrophobic proteins.

Growing a biofilm on a surface as a way of coating it, might seem like a bad idea, since there are quite a lot of coatings out there to prevent biofilms forming in the first place. But why not "fight fire with fire”, and create a biofilm that is non-pathogenic and prevents other biofouling from taking place.

Bacillus subtilis is an ideal candidate for a biofilm coating. Firstly because it is quickly grows a biofilm which has a smooth extracellular matrix. Secondly, the bacterium is a well known and extensively studied model organism which makes is easier to work with. Finally B. subtilis is a gram-positive bacterium like Streptomyces coelicolor, the bacterium that naturally produces hydrophobins. This might be an advantage when expressing and assembling the chaplin proteins in our host.


Biology


In nature, bacteria occur predominantly in highly organized multicellular communities called biofilms. Biofilm formation involves a complex developmental process, where cells differ from each other spatially and morphologically. The bacterial cells in biofilms are phenotypically different, demonstrating an intriguing example of heterogeneous regulation within an isogenic culture. Gram-positive bacteria have developed different strategies for survival in unfavorable environments, e.g. by getting competent or by sporulating. Biofilms offer an opportunity for the cells to survive extreme conditions as the cells in biofilms are more resistant to antibiotics and other harsh circumstances like physical stress, drought or competing organisms. Bacillus even forms highly complex biofilms with a large degree of structural complexity and diversification of cell function within the biofilm. There are even channels within the biofilm to allow drainage of waste and diffusion of oxygen deep within the biofilm.(Akos Kovacs)


===Biofilm formation===
Strain rok.jpg

Biofilm formation usually starts with the accumulation of biomass, next there is the adhesion to a surface by the production of adhesion proteins. Then the production of "extracellular polymeric substances" (EPS) starts and the phenotypic diversification. After maturation of the biofilm sporulation kicks in. Since the pathways involved in biofilm formation in B. subtilis are just starting to be unravelled, not everything is known about the complex physiological interactions within a biofilm. By using an already existing pathway in B. subtilis for the auto-induction of our hydrophobic proteins, we try to minimize the amount of tinkering to the existing signaling pathways. Thereby leaving the natural system intact.

Timing is one the most important factors in successful assembly of our chaplins in EPS. B. subtilis produces a protein that forms amyloidfibers called TasA. TasA is a very important protein to provide structural integrity in B. subtilis biofilms and is formed in the late stage of biofilm formation. The amyloid fibers that are formed provide the biofilm with an increased degree of rigidity (Romero et al, 2009). Chaplins also assemble into amyloid fibers and provide a similar function in the hyphae of S. coelicolor (Cleassen et al, 2009), giving the hyphae the structural ability to grow high up in the air. Incorperating the chaplins at the same moment as TasA is formed would maximize the chance of successful assembly of chaplins in the EPS, while enabling maximum biofilm coverage. For more details on our expression pathway check out our expression or modeling page.


Agar TY corn starch.jpg

=Coating surfaces

Prevention from our biofilm to grow out of control, is an important aspect when you would apply the hydrophobofilm outside the lab. To deal with these safety issues we modelled a kill switch for our hydrophobofilm. This kill switch relies on the production of a toxin and anti toxin. Where the anti toxin has a slightly shorter half-life than the toxin, thereby eventually resulting in the toxification of the cell itself. This toxification would occur after maturation of the biofilm. After the autotoxification the cells, the EPS with the embedded chaplin proteins will dry out, leaving a hydrophobic EPS layer on the surface.


Biofilm on ceramics.jpg

Applying our bacteria effectively to a surface poses big challenges. such as, how to coat a surface in a short period of time, with low cost and low tech methods. Furthermore there must be enough nutrients for the organisms to successfully form a biofilm, yet you do not want to smear you surface in to much medium, so to avoid that the organism will only adhere to the medium and not to the surface itself.


ceramics

Biofilm paste We attempted to make a medium that could be easily applied to a surface and enable biofilm formation to take place. To achieve this we tried to make our medium more viscous. By adding corn starch to regular TY medium we increased the viscosity of our medium and also made it richer in nutrients. We experimented with different corn starch concentrations.

We have created an easily applicable paste, to grow our biofilmcoating on all kinds of different surfaces. Another effect of the addition of cornstarch to the medium is an increased growing speed.


A & B: B. subtilis biofilms grown overnight on ceramics coated with the biofilm paste. C: B subtilis biofilms dried out over four days, after formation.


References

1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms

2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus subtilis Biolms

3. Dennis Claessen, Rick Rink, Wouter de Jong, et al, 2009, A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils