http://2010.igem.org/wiki/index.php?title=Special:Contributions/Ekkers&feed=atom&limit=50&target=Ekkers&year=&month=2010.igem.org - User contributions [en]2024-03-28T17:36:58ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/File:Boomsalecta.jpgFile:Boomsalecta.jpg2010-10-28T03:59:37Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/File:Stukieplukie.jpgFile:Stukieplukie.jpg2010-10-28T03:58:53Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/20_September_2010Team:Groningen/20 September 20102010-10-28T03:57:54Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
'''Week 29'''<br />
<br />
'''Expression test'''-''Peter & David''<br />
<br />
Succesfull in disrupted samples!<br />
<br />
'''Exression experiment'''<br />
<br />
<br><br />
<br />
'''Peter & David'''<br />
<br />
<br><br />
<br />
For this experiment, the following B. subtilis 168 strains were used:<br />
<br />
<pre><br />
<br />
<br />
<br />
</pre><br />
<br />
<br><br />
<br />
All cultures were grown overnight at 37 degrees Celsius in a shaker room, the appropriate antibiotics were used at all points in time during this experiment. <br />
<br />
<br><br />
<br />
Overnight cultures were used to dilute to a B. subtilis culture of 0,1 OD, these strains were divided into ‘’induced’’ and ‘’non-induced’’. Induction with 0,5% subtilin was done at a OD of 0,5 (approximately 2,5 hours after growth of the 0,1 culture started). <br />
<br />
<br><br />
<br />
After that the OD of the cultures was measured every .. hours.<br />
<br />
<br><br />
<br />
'''Sample preperation'''<br />
<br />
<br><br />
<br />
After .. hours, .. after induction, the samples were collected and processed. The following procedures were used:<br />
<br />
<br><br />
<br />
Pellet preperation ([[PelletPrepGR]])<br />
<br />
<br><br />
<br />
Supernatant processing ([[SupernatantPrepGR]])<br />
<br />
<br><br />
<br />
Cell disruption ([[ExtractionCellWallsGR]])<br />
<br />
<br><br />
<br />
Lysozyme preperation ([[LysozymePrepGR]])<br />
<br />
<br><br />
<br />
Analysis was done using SDS-PAGE ([[SDS-PAGEGR]]) and THT staining ([[THTstainingGR]]).<br />
<br />
<br><br />
<br />
'''Results''':<br />
<br />
<br><br />
<br />
'''Growth Curve'''<br />
<br />
<br><br />
<br />
[[Image:GrowthCurveGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
When preparing the pellets for disruption there was a difference visible between the induced and the non induced strains. The induced sample on the left formed a somewhat flaky pellet in comparison to the non induced sample.<br />
[[Image:pelletare weird.jpg]]<br />
<br />
<br><br />
<br />
'''THT Staining'''<br />
Disrupting are samples before ThT staining proved very succesful. Disruption isolates the cellenvelop fraction of the cells. And because the samples are cooked twice in SDS, a lot of proteins that would normally create noise in the ThT measurement are denaturized. These leaves only the persistant chaplin proteins in the sample. This was shown very well on the ThT emission below, with the induced samples showing a clear emission peak around the 482 nm just as is exspected with amyloids. The non-induced samples have such low emissions that it more or less equals the blanco. <br />
<br />
<br><br />
<br />
[[Image:eisuccesatlast.jpg|400px]]<br />
<br />
<br><br />
<br />
<br />
<br><br />
<br />
'''SDS-PAGE'''<br />
<br />
<br><br />
<br />
[[Image:SDS-PAGEGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
<br><br />
<br />
<br />
<br />
'''Chaplin ladder on gel'''-''Peter''<br />
<br />
<br> <br />
Starting with transformations GFP in bacillus<br />
<br><br />
<br />
'''Modellers:''' Find constants and parameters for gene expression model ''Laura, Djoke''<br />
<br />
Initial programming of gene expression model in Matlab. ''Laura''<br />
<br />
'''David'''<br />
<br />
biofilm paste<br />
<br><br />
<br />
- Medium pasta<br />
The goal was to get a paste like medium that could easily be applied to surfaces. <br />
First we tried to get this consistency by adding low amounts of agar to liquid medium, but because of the property of agar to be either liquid or solid with no fase in between this resulted in bad coating properties. Agar does not have a vicious consistency and therefore does not easily adhere to surfaces. By adding different concentrations of cornstarch flour to liquid LB medium, this vicious property could be attained. <br />
Experimentations were done with 50 ml liquid LB medium: 0.5, 1, 2, 5 g corn starch flour. After adding the corn starch flour to the medium it was autoclaved. It seemed that the most optimal consistency was attained at 2 g per 50 ml, Creating a still 'pourable' liquid yet being very viscous. After cooling down, the paste further thickens.<br />
This 2g:50 ml was tested with bacillus strains: degU, Rok, <br />
<br />
[[Image:stukieplukie.jpg]]<br />
<br />
[[Image:boomsalecta.jpg]]<br />
<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>Ekkershttp://2010.igem.org/Team:Groningen/18_October_2010Team:Groningen/18 October 20102010-10-28T03:54:57Z<p>Ekkers: </p>
<hr />
<div>'''Modellers:''' Find constants and parameters for gene expression model, finish modelling and put everything on the wiki ''Laura, Djoke''<br />
<br />
Write wiki text for killswitch and gene expression model. Create all figures, tables and equations in wiki format for gene expression model and killswitch model. ''Laura''<br />
<br />
<br><br />
<br />
'''Peter en David'''<br />
<br />
Expression experiment contruct E&H chaplin and C chaplin. Growing in batches of 0.5 L liquid TY medium at 37 degrees. Inoculation with overnight culture with a starting OD of 0.1. Inducing at an OD of 0.5 with 1.5% subtilin overnight. Harvesting the overnight culture and washing pellet before disrupting it.<br />
<br />
THT measurement<br />
[[Image:C CN EH EHN gr.jpg]] <br />
<br><br />
Mass spec malditov results<br />
<br><br />
[[Image:massspec malditov disruption]]<br />
<br><br />
<br />
'''David'''<br />
<br />
measurements in Bremen on surface activity of chaplin coated objects<br />
<br />
We wanted to know if there was an effect of our chaplin proteins on surface drag in the water. To test this we wanted to measure the water resistance of a spindel shaped object in the water at different speeds. First do the measurements with a clean spindel and secondly after coating the spindel with purified chaplins from streptomyces. The spindle that was used for the mesurements, this shapes is designed minimize the effect of shape of the objects drag. Thereby maximizing the effect of surface resistance on the drag coeffient.<br />
<br />
<br />
[[Image:pinnacleunitvandennis.jpg]] <br />
<br />
<br></div>Ekkershttp://2010.igem.org/File:Pinnacleunitvandennis.jpgFile:Pinnacleunitvandennis.jpg2010-10-28T03:35:28Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/18_October_2010Team:Groningen/18 October 20102010-10-28T03:34:11Z<p>Ekkers: </p>
<hr />
<div>'''Modellers:''' Find constants and parameters for gene expression model, finish modelling and put everything on the wiki ''Laura, Djoke''<br />
<br />
Write wiki text for killswitch and gene expression model. Create all figures, tables and equations in wiki format for gene expression model and killswitch model. ''Laura''<br />
<br />
<br><br />
<br />
'''Peter en David'''<br />
<br />
Expression experiment contruct E&H chaplin and C chaplin. Growing in batches of 0.5 L liquid TY medium at 37 degrees. Inoculation with overnight culture with a starting OD of 0.1. Inducing at an OD of 0.5 with 1.5% subtilin overnight. Harvesting the overnight culture and washing pellet before disrupting it.<br />
<br />
THT measurement<br />
[[Image:C CN EH EHN gr.jpg]] <br />
<br><br />
Mass spec malditov results<br />
<br><br />
[[Image:massspec malditov disruption]]<br />
<br><br />
<br />
'''David'''<br />
<br />
measurements in Bremen on surface activity of chaplin coated objects<br />
The spindle that was used for the mesurements, this shapes is designed minimize the effect of shape of the objects drag. Thereby maximizing the effect of surface resistance on the drag coeffient.<br />
[[Image:pinnacleunitvandennis.jpg]] <br />
<br />
<br></div>Ekkershttp://2010.igem.org/Team:Groningen/20_September_2010Team:Groningen/20 September 20102010-10-28T03:17:11Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
'''Week 29'''<br />
<br />
'''Expression test'''-''Peter & David''<br />
<br />
Succesfull in disrupted samples!<br />
<br />
'''Exression experiment'''<br />
<br />
<br><br />
<br />
'''Peter & David'''<br />
<br />
<br><br />
<br />
For this experiment, the following B. subtilis 168 strains were used:<br />
<br />
<pre><br />
<br />
<br />
<br />
</pre><br />
<br />
<br><br />
<br />
All cultures were grown overnight at 37 degrees Celsius in a shaker room, the appropriate antibiotics were used at all points in time during this experiment. <br />
<br />
<br><br />
<br />
Overnight cultures were used to dilute to a B. subtilis culture of 0,1 OD, these strains were divided into ‘’induced’’ and ‘’non-induced’’. Induction with 0,5% subtilin was done at a OD of 0,5 (approximately 2,5 hours after growth of the 0,1 culture started). <br />
<br />
<br><br />
<br />
After that the OD of the cultures was measured every .. hours.<br />
<br />
<br><br />
<br />
'''Sample preperation'''<br />
<br />
<br><br />
<br />
After .. hours, .. after induction, the samples were collected and processed. The following procedures were used:<br />
<br />
<br><br />
<br />
Pellet preperation ([[PelletPrepGR]])<br />
<br />
<br><br />
<br />
Supernatant processing ([[SupernatantPrepGR]])<br />
<br />
<br><br />
<br />
Cell disruption ([[ExtractionCellWallsGR]])<br />
<br />
<br><br />
<br />
Lysozyme preperation ([[LysozymePrepGR]])<br />
<br />
<br><br />
<br />
Analysis was done using SDS-PAGE ([[SDS-PAGEGR]]) and THT staining ([[THTstainingGR]]).<br />
<br />
<br><br />
<br />
'''Results''':<br />
<br />
<br><br />
<br />
'''Growth Curve'''<br />
<br />
<br><br />
<br />
[[Image:GrowthCurveGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
When preparing the pellets for disruption there was a difference visible between the induced and the non induced strains. The induced sample on the left formed a somewhat flaky pellet in comparison to the non induced sample.<br />
[[Image:pelletare weird.jpg]]<br />
<br />
<br><br />
<br />
'''THT Staining'''<br />
Disrupting are samples before ThT staining proved very succesful. Disruption isolates the cellenvelop fraction of the cells. And because the samples are cooked twice in SDS, a lot of proteins that would normally create noise in the ThT measurement are denaturized. These leaves only the persistant chaplin proteins in the sample. This was shown very well on the ThT emission below, with the induced samples showing a clear emission peak around the 482 nm just as is exspected with amyloids. The non-induced samples have such low emissions that it more or less equals the blanco. <br />
<br />
<br><br />
<br />
[[Image:eisuccesatlast.jpg|400px]]<br />
<br />
<br><br />
<br />
<br />
<br><br />
<br />
'''SDS-PAGE'''<br />
<br />
<br><br />
<br />
[[Image:SDS-PAGEGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
<br><br />
<br />
<br />
<br />
'''Chaplin ladder on gel'''-''Peter''<br />
<br />
<br> <br />
Starting with transformations GFP in bacillus<br />
<br><br />
<br />
'''Modellers:''' Find constants and parameters for gene expression model ''Laura, Djoke''<br />
<br />
Initial programming of gene expression model in Matlab. ''Laura''<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>Ekkershttp://2010.igem.org/Team:Groningen/20_September_2010Team:Groningen/20 September 20102010-10-28T02:49:58Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
'''Week 29'''<br />
<br />
'''Expression test'''-''Peter & David''<br />
<br />
Succesfull in disrupted samples!<br />
<br />
'''Exression experiment'''<br />
<br />
<br><br />
<br />
'''Peter & David'''<br />
<br />
<br><br />
<br />
For this experiment, the following B. subtilis 168 strains were used:<br />
<br />
<pre><br />
<br />
<br />
<br />
</pre><br />
<br />
<br><br />
<br />
All cultures were grown overnight at 37 degrees Celsius in a shaker room, the appropriate antibiotics were used at all points in time during this experiment. <br />
<br />
<br><br />
<br />
Overnight cultures were used to dilute to a B. subtilis culture of 0,1 OD, these strains were divided into ‘’induced’’ and ‘’non-induced’’. Induction with 0,5% subtilin was done at a OD of 0,5 (approximately 2,5 hours after growth of the 0,1 culture started). <br />
<br />
<br><br />
<br />
After that the OD of the cultures was measured every .. hours.<br />
<br />
<br><br />
<br />
'''Sample preperation'''<br />
<br />
<br><br />
<br />
After .. hours, .. after induction, the samples were collected and processed. The following procedures were used:<br />
<br />
<br><br />
<br />
Pellet preperation ([[PelletPrepGR]])<br />
<br />
<br><br />
<br />
Supernatant processing ([[SupernatantPrepGR]])<br />
<br />
<br><br />
<br />
Cell disruption ([[ExtractionCellWallsGR]])<br />
<br />
<br><br />
<br />
Lysozyme preperation ([[LysozymePrepGR]])<br />
<br />
<br><br />
<br />
Analysis was done using SDS-PAGE ([[SDS-PAGEGR]]) and THT staining ([[THTstainingGR]]).<br />
<br />
<br><br />
<br />
'''Results''':<br />
<br />
<br><br />
<br />
'''Growth Curve'''<br />
<br />
<br><br />
<br />
[[Image:GrowthCurveGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
When preparing the pellets for disruption there was a difference visible between the induced and the non induced strains. The induced sample on the left formed a somewhat flaky pellet in comparison to the non induced sample.<br />
[[Image:pelletare weird.jpg]]<br />
<br />
<br><br />
<br />
'''THT Staining'''<br />
<br />
<br><br />
<br />
[[Image:eisuccesatlast.jpg|400px]]<br />
<br />
<br><br />
<br />
<br />
<br><br />
<br />
'''SDS-PAGE'''<br />
<br />
<br><br />
<br />
[[Image:SDS-PAGEGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
<br><br />
<br />
<br />
<br />
'''Chaplin ladder on gel'''-''Peter''<br />
<br />
<br> <br />
Starting with transformations GFP in bacillus<br />
<br><br />
<br />
'''Modellers:''' Find constants and parameters for gene expression model ''Laura, Djoke''<br />
<br />
Initial programming of gene expression model in Matlab. ''Laura''<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>Ekkershttp://2010.igem.org/File:Pelletare_weird.jpgFile:Pelletare weird.jpg2010-10-28T02:47:51Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/20_September_2010Team:Groningen/20 September 20102010-10-28T02:46:32Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
'''Week 29'''<br />
<br />
'''Expression test'''-''Peter & David''<br />
<br />
Succesfull in disrupted samples!<br />
<br />
'''Exression experiment'''<br />
<br />
<br><br />
<br />
'''Peter & David'''<br />
<br />
<br><br />
<br />
For this experiment, the following B. subtilis 168 strains were used:<br />
<br />
<pre><br />
<br />
<br />
<br />
</pre><br />
<br />
<br><br />
<br />
All cultures were grown overnight at 37 degrees Celsius in a shaker room, the appropriate antibiotics were used at all points in time during this experiment. <br />
<br />
<br><br />
<br />
Overnight cultures were used to dilute to a B. subtilis culture of 0,1 OD, these strains were divided into ‘’induced’’ and ‘’non-induced’’. Induction with 0,5% subtilin was done at a OD of 0,5 (approximately 2,5 hours after growth of the 0,1 culture started). <br />
<br />
<br><br />
<br />
After that the OD of the cultures was measured every .. hours.<br />
<br />
<br><br />
<br />
'''Sample preperation'''<br />
<br />
<br><br />
<br />
After .. hours, .. after induction, the samples were collected and processed. The following procedures were used:<br />
<br />
<br><br />
<br />
Pellet preperation ([[PelletPrepGR]])<br />
<br />
<br><br />
<br />
Supernatant processing ([[SupernatantPrepGR]])<br />
<br />
<br><br />
<br />
Cell disruption ([[ExtractionCellWallsGR]])<br />
<br />
<br><br />
<br />
Lysozyme preperation ([[LysozymePrepGR]])<br />
<br />
<br><br />
<br />
Analysis was done using SDS-PAGE ([[SDS-PAGEGR]]) and THT staining ([[THTstainingGR]]).<br />
<br />
<br><br />
<br />
'''Results''':<br />
<br />
<br><br />
<br />
'''Growth Curve'''<br />
<br />
<br><br />
<br />
[[Image:GrowthCurveGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
When preparing the pellets for disruption there was a difference visible between the induced and the non induced strains. The induced sample on the left formed a somewhat flaky pellet in comparison to the non induced sample.<br />
[[Image:pelletare weird.jpg]]<br />
<br />
<br><br />
<br />
'''THT Staining'''<br />
<br />
<br><br />
<br />
[[Image:eisuccesatlast.jpg|400px]]<br />
<br />
<br><br />
<br />
<br />
<br><br />
<br />
'''SDS-PAGE'''<br />
<br />
<br><br />
<br />
[[Image:SDS-PAGEGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
<br><br />
<br />
<br />
<br />
'''Chaplin ladder on gel'''-''Peter''<br />
<br />
'''Modellers:''' Find constants and parameters for gene expression model ''Laura, Djoke''<br />
<br />
Initial programming of gene expression model in Matlab. ''Laura''<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>Ekkershttp://2010.igem.org/File:Eisuccesatlast.jpgFile:Eisuccesatlast.jpg2010-10-28T02:39:09Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/20_September_2010Team:Groningen/20 September 20102010-10-28T02:32:20Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
'''Week 29'''<br />
<br />
'''Expression test'''-''Peter & David''<br />
<br />
Succesfull in disrupted samples!<br />
<br />
'''Exression experiment'''<br />
<br />
<br><br />
<br />
'''Peter & David'''<br />
<br />
<br><br />
<br />
For this experiment, the following B. subtilis 168 strains were used:<br />
<br />
<pre><br />
<br />
<br />
<br />
</pre><br />
<br />
<br><br />
<br />
All cultures were grown overnight at 37 degrees Celsius in a shaker room, the appropriate antibiotics were used at all points in time during this experiment. <br />
<br />
<br><br />
<br />
Overnight cultures were used to dilute to a B. subtilis culture of 0,1 OD, these strains were divided into ‘’induced’’ and ‘’non-induced’’. Induction with 0,5% subtilin was done at a OD of 0,5 (approximately 2,5 hours after growth of the 0,1 culture started). <br />
<br />
<br><br />
<br />
After that the OD of the cultures was measured every .. hours.<br />
<br />
<br><br />
<br />
'''Sample preperation'''<br />
<br />
<br><br />
<br />
After .. hours, .. after induction, the samples were collected and processed. The following procedures were used:<br />
<br />
<br><br />
<br />
Pellet preperation ([[PelletPrepGR]])<br />
<br />
<br><br />
<br />
Supernatant processing ([[SupernatantPrepGR]])<br />
<br />
<br><br />
<br />
Cell disruption ([[ExtractionCellWallsGR]])<br />
<br />
<br><br />
<br />
Lysozyme preperation ([[LysozymePrepGR]])<br />
<br />
<br><br />
<br />
Analysis was done using SDS-PAGE ([[SDS-PAGEGR]]) and THT staining ([[THTstainingGR]]).<br />
<br />
<br><br />
<br />
'''Results''':<br />
<br />
<br><br />
<br />
'''Growth Curve'''<br />
<br />
<br><br />
<br />
[[Image:GrowthCurveGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
<br />
<br />
<br><br />
<br />
'''THT Staining'''<br />
<br />
<br><br />
<br />
[[Image:eisuccesatlast.jpg|400px]]<br />
<br />
<br><br />
<br />
<br />
<br><br />
<br />
'''SDS-PAGE'''<br />
<br />
<br><br />
<br />
[[Image:SDS-PAGEGR6.jpg|400px]]<br />
<br />
<br><br />
<br />
<br><br />
<br />
<br />
<br />
'''Chaplin ladder on gel'''-''Peter''<br />
<br />
'''Modellers:''' Find constants and parameters for gene expression model ''Laura, Djoke''<br />
<br />
Initial programming of gene expression model in Matlab. ''Laura''<br />
<br />
<br />
<br />
<br />
<br />
{{Team:Groningen/Footer}}</div>Ekkershttp://2010.igem.org/Team:Groningen/26_July_2010Team:Groningen/26 July 20102010-10-28T02:30:02Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
<br />
<br />
'''Week 30'''<br />
----<br />
<br />
<br />
'''Jorrit'''<br />
<br />
Tested expression of week 29 on SDS-page with silver staining on the CFE, medium & pellet samples, results were inconclusive.<br />
<br />
<br />
Transformation ''B sub.'' (according to [https://2010.igem.org/Team:Groningen/Protocols_for_Bacillus_subtilis_168 protocol])<br />
<br />
Inoculated NZ8900 ΔTasA in 10 ml MM, overnight @ 37°C shaker.<br />
<br />
1 ml overnight culture NZ8900 ΔTasA in 10 ml fresh MM for 3 hours @ 37°C shaker.<br />
<br />
Added approx. 1 µg plasmid E and H in two 2 ml tubes and incubated 30 min @ 37°C shaker.<br />
<br />
Diluted in pre warmed TY (300 µl) and put for 45 min @ 37°C shaker.<br />
<br />
Plated on LB-agar with antibiotic Cm5/Spec100 and grown @ 37°C (colonies @ 30/7).<br />
<br />
<br />
Inoculated 4 H colonies and 4 E colonies in 5 ml TY with antibiotics Km5 ,Cm5 and Spec100 and put overnight @ 37°C shaker.<br />
<br />
Two -80°C stocks were made of ''B. sub'' NZ8900 ΔTasA with pNZ8901_E and B. sub NZ8900 ΔTasA with pNZ8901_H.<br />
<br />
<br />
Chaplin expression (according to [https://2010.igem.org/Team:Groningen/Chaplins protocol])<br />
<br />
Inoculated 4 Greiner tubes with pNZ9801 E6, E2, H1, H3 in 5 ml TY with antibiotic Km5 and Cm5.<br />
<br />
10 µl of OD cultures (pNZ9801 E6, E2, H1, H3) was taken and grown , overnight @ 37°C shaker, in 5 ml TY with antibiotic Cm5/Spec100 and Cm5.<br />
<br />
Done growth curve with OD- measurement.<br />
<br />
Of all measurementperiodes were taken 2 ml samples, spinned down and put supernatant and cell pellet in freezer.<br />
<br />
<br />
'''Modellers:''':<br />
<br />
We read and looked up some more articles on biofilm formation models. We found that Professor Van Loosdrecht in Delft knows a lot about this and planned a meeting with him. ''Joël, Arend, Laura, Djoke''<br />
<br />
<br />
'''Arend Jan'''<br />
<br />
<br />
The PCR product from the insertion of Biobrick sites into the pNZ8048 plasmid was cleaned, digested with BamHI, and cleaned again. After cleanup the concentration was to low to continue with a ligation. The cleaned PCR product was used again for a digestion with BamHI and this time the enzyme was then heat inactivated (30min. 80C). To this mixture a 20X ATP solution was added and T4 ligase. After ligation the mixture was transformed to E.coli TOP10. Like before, the plasmid won’t be transformed to E.coli for some reason. The leftover ligation mixture was desalted by use of an osmotic filter and electroporated to Lactococcus lactis (Because the plasmid is originally a lactis plasmid). This also did not work. Should ask if the transformation was done correctly. If so, then go over the cloning strategy again... stupid plasmid.<br />
<br />
<br />
At the same time we’re still trying to get rid of the IS element. Like before a restriction with MunI and XhoI was performed, this time with both enzymes from Fermentas, on the plasmids pNZ8901-bbs and pNZ8048.<br />
<br />
<br />
[[Image:28-07-10gn.jpg|150px|thumb|left|pNZ8084 in left lane, pNZ8901-bbs in middle lane.]]<br />
<br style="clear: both" /><br />
<br />
The fragments indicate that XhoI did not cut in the used buffer(G), while it should have 50-100% activity. Not continuing with restrictions but focusing on PCR to get rid of IS element.<br />
<br />
<br />
<br />
'''Geeske'''<br />
<br />
Vacation in Berlin, had fun there!<br />
<br />
'''David'''<br />
<br />
Test purified chaplins <br />
Testing surface tension reduction, by monomerizing 5 mg chaplins via TFA treatment. Then redilute chaplins in 500 ul demiwater an put a drop 50 ul on a petridish and leaf it to dry out overnight.<br />
Next day pipet 50 ul water on to the dried in chaplins and on a clean petri dish and compare.<br />
<br><br />
[[Image:petrichaplins.jpg]]<br />
<br />
{{Team:Groningen/Footer}}</div>Ekkershttp://2010.igem.org/File:Ceramic_coating_gr1.jpgFile:Ceramic coating gr1.jpg2010-10-28T02:18:20Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/File:Disrupted_last_measurement_gr.jpgFile:Disrupted last measurement gr.jpg2010-10-28T02:06:54Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/25_October_2010Team:Groningen/25 October 20102010-10-28T02:02:57Z<p>Ekkers: </p>
<hr />
<div>'''David, Arend Jan and Peter'''<br />
<br />
Growing transformants with GFP labeled SrfA and yqxM promotors. To see when the cell density dependant quorum sensing system kicks in to activate the promotors, sequential cultures were inoculated every our at an OD of 0.1. After 7 hours, the OD from the sequential cultures was measured together with the GFP fluorescence.<br />
<br />
GFP fluorence:<br />
<br />
OD mesearements:<br />
<br />
'''David'''<br />
<br><br />
Biofilm paste experiment <br />
<br> <br />
Growing biofilms on sterilized ceramics that have been coated with athin layer of TY medium with corn starch.<br />
<br><br />
Mix: 30 mg cornstarch/ml TY 1% agar medium and 0.5% glucose<br />
<br />
After dissolving the agar and addition of corn starch teh pieces of cermics we added an taken out <br />
<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
<br />
<br />
Expression experiment: <br />
<br />
[[Image:growthchaplineEN.jpg]]<br />
<br />
ThT measurement of EH induced together with C non induced(this due to a mixup during he experiment), EH non induced, C induced<br />
<br />
[[Image:disrupted last measurement gr.jpg]]</div>Ekkershttp://2010.igem.org/Team:Groningen/11_October_2010Team:Groningen/11 October 20102010-10-28T02:02:10Z<p>Ekkers: </p>
<hr />
<div>'''Laura'''<br />
<br />
Debugging the matlab model for gene expression and killswitch. Find more information about constants used in the gene expression model.<br />
<br />
'''Peter & David'''<br />
<br><br />
Expression experiment on new transformants: E+H1, E+H2, C1, C2, E and H mix 4 clones.<br />
Growing the strains from overnight cultures in 5 ml of TY medium at 37 degrees.<br />
<br />
'''David'''<br />
<br><br />
Biofilm paste experiment <br />
<br> <br />
Growing biofilms on sterilized ceramics that have been coated with athin layer of TY medium with corn starch.<br />
<br><br />
Mix: 30 mg cornstarch/ml TY 1% agar medium and 0.5% glucose<br />
<br />
After dissolving the agar and addition of corn starch teh pieces of cermics we added an taken out <br />
<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
<br />
<br />
Expression experiment: <br />
<br />
[[Image:growthchaplineEN.jpg]]<br />
<br />
ThT measurement of EH induced together with C non induced(this due to a mixup during he experiment), EH non induced, C induced<br />
<br />
[[Image:disrupted last measurement gr.jpg]]</div>Ekkershttp://2010.igem.org/Team:Groningen/27_September_2010Team:Groningen/27 September 20102010-10-28T01:59:11Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
'''Week 29'''<br />
<br />
'''Chaplin ladder on gel'''-''Peter''<br />
<br />
<br />
The Yqxm and the Srfa (both variants)promoters where extracted from genomic ''Bacillus Subtilis'' DNA via PCR and inserted into the PNZ8901 plasmid. All transformations where done in ''Lactococcus Lactis''.<br />
Restriction analysis was performed to check the constructs. The concentrations where determined with the Nanodrop<br />
<br />
<br />
{| border="1"<br />
| <br />
| Srfa 1<br />
| Srfa 2<br />
| Yqxm<br />
|-<br />
| Clone 1<br />
| 75.3mg/ml<br />
| 78.9mg/ml<br />
| 72.0mg/mL<br />
|-<br />
| Clone 2<br />
| 25.6mg/ml<br />
| 40.2mg/ml<br />
| 57.6mg/mL<br />
|-<br />
| Clone 3<br />
| 44.4mg/ml<br />
| False positive<br />
| False positive<br />
|}<br />
<br />
<br />
'''Laura'''<br />
<br />
Debugging the matlab model of killswitch and gene expression.<br />
{{Team:Groningen/Footer}}<br />
<br />
'''David'''<br />
<br><br />
Biofilm paste experiment <br />
<br> <br />
Growing biofilms on sterilized ceramics that have been coated with athin layer of TY medium with corn starch.<br />
<br><br />
Mix: 30 mg cornstarch/ml TY 1% agar medium and 0.5% glucose<br />
<br />
After dissolving the agar and addition of corn starch teh pieces of cermics we added an taken out <br />
<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
<br />
<br />
Expression experiment: <br />
<br />
[[Image:growthchaplineEN.jpg]]<br />
<br />
ThT measurement of EH induced together with C non induced(this due to a mixup during he experiment), EH non induced, C induced<br />
<br />
[[Image:disrupted last measurement gr.jpg]]</div>Ekkershttp://2010.igem.org/Team:Groningen/27_September_2010Team:Groningen/27 September 20102010-10-28T01:34:01Z<p>Ekkers: </p>
<hr />
<div>{{Team:Groningen/Header}}<br />
<br />
'''Week 29'''<br />
<br />
'''Chaplin ladder on gel'''-''Peter''<br />
<br />
<br />
The Yqxm and the Srfa (both variants)promoters where extracted from genomic ''Bacillus Subtilis'' DNA via PCR and inserted into the PNZ8901 plasmid. All transformations where done in ''Lactococcus Lactis''.<br />
Restriction analysis was performed to check the constructs. The concentrations where determined with the Nanodrop<br />
<br />
<br />
{| border="1"<br />
| <br />
| Srfa 1<br />
| Srfa 2<br />
| Yqxm<br />
|-<br />
| Clone 1<br />
| 75.3mg/ml<br />
| 78.9mg/ml<br />
| 72.0mg/mL<br />
|-<br />
| Clone 2<br />
| 25.6mg/ml<br />
| 40.2mg/ml<br />
| 57.6mg/mL<br />
|-<br />
| Clone 3<br />
| 44.4mg/ml<br />
| False positive<br />
| False positive<br />
|}<br />
<br />
<br />
'''Laura'''<br />
<br />
Debugging the matlab model of killswitch and gene expression.<br />
{{Team:Groningen/Footer}}<br />
<br />
'''David'''<br />
<br><br />
Biofilm paste experiment <br />
<br> <br />
Growing biofilms on sterilized ceramics that have been coated with athin layer of TY medium with corn starch.<br />
<br><br />
Mix: 30 mg cornstarch/ml TY 1% agar medium and 0.5% glucose<br />
<br />
After dissolving the agar and addition of corn starch <br />
<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]<br />
[[Image:ceramic coating gr1.jpg]]</div>Ekkershttp://2010.igem.org/Team:Groningen/25_October_2010Team:Groningen/25 October 20102010-10-28T01:12:58Z<p>Ekkers: New page: '''David, Arend Jan and Peter''' Growing transformants with GFP labeled SrfA and yqxM promotors. To see when the cell density dependant quorum sensing system kicks in to activate the prom...</p>
<hr />
<div>'''David, Arend Jan and Peter'''<br />
<br />
Growing transformants with GFP labeled SrfA and yqxM promotors. To see when the cell density dependant quorum sensing system kicks in to activate the promotors, sequential cultures were inoculated every our at an OD of 0.1. After 7 hours, the OD from the sequential cultures was measured together with the GFP fluorescence.<br />
<br />
GFP fluorence:<br />
<br />
OD mesearements:</div>Ekkershttp://2010.igem.org/Team:Groningen/11_October_2010Team:Groningen/11 October 20102010-10-28T01:01:20Z<p>Ekkers: </p>
<hr />
<div>'''Laura'''<br />
<br />
Debugging the matlab model for gene expression and killswitch. Find more information about constants used in the gene expression model.<br />
<br />
'''Peter & David'''<br />
<br><br />
Expression experiment on new transformants: E+H1, E+H2, C1, C2, E and H mix 4 clones.<br />
Growing the strains from overnight cultures in 5 ml of TY medium at 37 degrees</div>Ekkershttp://2010.igem.org/Team:Groningen/18_October_2010Team:Groningen/18 October 20102010-10-28T00:51:26Z<p>Ekkers: </p>
<hr />
<div>'''Modellers:''' Find constants and parameters for gene expression model, finish modelling and put everything on the wiki ''Laura, Djoke''<br />
<br />
Write wiki text for killswitch and gene expression model. Create all figures, tables and equations in wiki format for gene expression model and killswitch model. ''Laura''<br />
<br />
<br><br />
<br />
'''Peter en David'''<br />
<br />
Expression experiment contruct E&H chaplin and C chaplin. Growing in batches of 0.5 L liquid TY medium at 37 degrees. Inoculation with overnight culture with a starting OD of 0.1. Inducing at an OD of 0.5 with 1.5% subtilin overnight. Harvesting the overnight culture and washing pellet before disrupting it.<br />
<br />
THT measurement<br />
[[Image:C CN EH EHN gr.jpg]] <br />
<br><br />
Mass spec malditov results<br />
<br><br />
[[Image:massspec malditov disruption]]<br />
<br><br />
<br />
'''David'''<br />
<br />
measurements in Bremen on surface activity of chaplin coated objects<br />
<br />
<br></div>Ekkershttp://2010.igem.org/Team:Groningen/ApplicationsTeam:Groningen/Applications2010-10-28T00:19:05Z<p>Ekkers: /* Anti-fouling coatings */</p>
<hr />
<div>__NOTOC__<br />
==Applications==<br />
A biofilm coating can be used for a great number of applications. We developed our hydrophobic coating keeping in mind that future development of our project could focus on adding other properties to a biofilm coating as well, creating functional coatings with different purposes. Utilizing the qualities of different biofilm forming bacteria or micro organisms and combining these coating properties with the ever growing stack of biobricks within the database could give rise to a variety of engineered biofilm machines, capable of transforming surfaces under many conditions.<br />
<br />
===Hydrophobic coatings===<br />
The potential benefits of hydrophobic coatings can be put to use in a wide variety of application areas; hydrophobic clothes, anti-fouling coatings, anti-corrosion coatings, hydrophobic paint and medical coatings for drug delivery or against biofilm formation. For these applications a lot of hydrophobic coatings have been developed. Not all of these coatings have been succesfully used outside the lab, so the search for a cheap, multi-functional hydrophobic coating is still ongoing.<br />
<br />
<br><br />
<br />
===Anti-fouling coatings===<br />
Surface hydrophobicity could also be beneficial in the antifouling industry, for instance when applied on ships. When marine micro-organisms like algae or pocks adhere to the hull of ships, they form a layer which greatly increases drag in the water. This results in higher fuel costs and increased erosion. To prevent organisms to adhere to the hull of ships, chemical antifouling paints, which often contain copper and tin, are used. 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 tons of organotin and 30 tons of copper end up in the environment every year. With our biological coating this impact on the environment could be greatly reduced.<br />
<br />
<br><br />
<br />
'''Must be''' <br />
<br>Anticorrosive <br />
<br>Antifouling <br />
<br>Environmentally acceptable <br />
<br>Economically viable <br />
<br>Long life <br />
<br>A target for non-specific species<br />
<br>Compatible with underlying system<br />
<br>Resistant to abrasion/biodegradation/erosion<br />
<br>Capable of protecting regardless of operational profile<br />
<br>Smooth<br />
<br />
<br><br />
<br />
'''Must not be:'''<br />
<br>Toxic to the environment<br />
<br>Persistent in the environment<br />
<br>Expensive<br />
<br>Chemically unstable<br />
<br>A target for non-specific species<br />
<br />
<br><br />
<br />
Organotin pollution in China: an overview of the current state and potential health risk - Cao et al.<br />
<br />
===Biofilm coatings===<br />
<br />
Although a biofilm is not ideal for all of these coating purposes (imagine a biofilm jacket), an hydrophobic biofilm coating could be applied in fields ranging from anti-fouling coatings on ships, peers and buoys, to coatings used to protect catheters and protheses from pathogenic bacteria. Moreover, with introducing our kill switch and our chaplin coating without living bacteria, a even broader range of applications can be taken into consideration as certain ethicial and safety issues are dealth with.<br />
<br />
<br><br />
The main advantage of a biofilm coating is that it is very cheap, applying it requires no technical pinnacles and it is more environmentally friendly than certain chemical coatings. Next to that, biofilms can grow on a wide variety of surfaces: They are found on your teeth, in catheters, in plumbing, in water cleaning installations (beneficial), in bioreactors and, if you're one of those students, on your bathroom floor (blech).<br />
<br />
<br><br />
So far there have been coatings with biological substances, but bacteria where only used to produce the coating material. In our project the bacteria will form a biofilm on the desired surface, which will then function as a coating. Our bacteria therefore execute the coating process themselves, which could save a lot of effort. In the case of using chaplins as a building blocks for a hydrophobic coating, the biofilm is also used to orient and anchor the proteins in the right way. As chaplins are amphipathic, the orientation in their pure form is determined by the properties of the surface they coat as well: Using a biofilm to structure them the right way is an easy and smart solution that helps us to get around some problems of using the chaplins in their pure form as a coating.<br />
<br />
<br><br />
<br />
===Medical coatings===<br />
Because of their surface modifying abilities, hydrophobic biofilms of none-pathogenic bacteria may be used to prevent pathogenic biofilms from adhering to prothesis or catheters. Keeping in mind that growing a biofilm, even our ''good'' biofilm coating, in a catheter or on prothesis can give serious medical problems like shock or inflammation. Therefore extensive research would need to be done before our this would be applicable. ''Bacillus subtilis'' is not pathogenic but if needed another bacterium could be used to form the hydrophobic biofilm like ''Lactococcus'', a natural inhabitant of our body. The principle via which we form a ''Bacillus'' biofilm coating is applicable to a number of hosts. Next to that, research could be done to perhaps alter the chaplins so the coating could be functionalized for drug delivery, cell adhesion or anti-bacterial activity.<br />
<br><br />
<br />
===Concrete protection===<br />
As the [https://2010.igem.org/Team:Newcastle Newcastle] team enters this years’ competition with a ''B. subtilis'' machine that fixes cracks in concrete, preventing corrosion and water- or frost-induced damage to buildings, roads or monuments; we have a great addition to their project (or the other way around). Hydrophobic coatings are used to protect concrete structures from corrosion damage, especially when reinforced with metal bars. Our ''B. subtilis'' biofilm would be able to do the same thing by filling up cracks and reducing water inflow by chaplin production: thus preventing damage by water or ice.<br />
<br />
<br><br />
A combination of both projects would make for great bacterial machinery: As the crack-filling bacteria repel the water and ice that is damaging concrete structures further damage is prevented and repair is accelerated.</div>Ekkershttp://2010.igem.org/Team:Groningen/ApplicationsTeam:Groningen/Applications2010-10-28T00:17:24Z<p>Ekkers: /* Anti-fouling coatings */</p>
<hr />
<div>__NOTOC__<br />
==Applications==<br />
A biofilm coating can be used for a great number of applications. We developed our hydrophobic coating keeping in mind that future development of our project could focus on adding other properties to a biofilm coating as well, creating functional coatings with different purposes. Utilizing the qualities of different biofilm forming bacteria or micro organisms and combining these coating properties with the ever growing stack of biobricks within the database could give rise to a variety of engineered biofilm machines, capable of transforming surfaces under many conditions.<br />
<br />
===Hydrophobic coatings===<br />
The potential benefits of hydrophobic coatings can be put to use in a wide variety of application areas; hydrophobic clothes, anti-fouling coatings, anti-corrosion coatings, hydrophobic paint and medical coatings for drug delivery or against biofilm formation. For these applications a lot of hydrophobic coatings have been developed. Not all of these coatings have been succesfully used outside the lab, so the search for a cheap, multi-functional hydrophobic coating is still ongoing.<br />
<br />
<br><br />
<br />
===Anti-fouling coatings===<br />
Surface hydrophobicity could also be beneficial in the antifouling industry, for instance when applied on ships. When marine micro-organisms like algae or pocks adhere to the hull of ships, they form a layer which greatly increases drag in the water. This results in higher fuel costs and increased erosion. To prevent organisms to adhere to the hull of ships, chemical antifouling paints, which often contain copper and tin, are used. 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 tons of organotin and 30 tons of copper end up in the environment every year. With our biological coating this impact on the environment could be greatly reduced.<br />
<br />
<br><br />
<br />
'''Must be''' <br />
<br>Anticorrosive <br />
<br>Antifouling <br />
<br>Environmentally acceptable <br />
<br>Economically viable <br />
<br>Long life <br />
<br>A target for non-specific species<br />
<br>Compatible with underlying system<br />
<br>Resistant to abrasion/biodegradation/erosion<br />
<br>Capable of protecting regardless of operational profile<br />
<br>Smooth<br />
<br />
<br><br />
<br />
'''Must not be:'''<br />
Toxic to the environment<br />
Persistent in the environment<br />
Expensive<br />
Chemically unstable<br />
A target for non-specific species<br />
<br />
<br><br />
<br />
Organotin pollution in China: an overview of the current state and potential health risk - Cao et al.<br />
<br />
===Biofilm coatings===<br />
<br />
Although a biofilm is not ideal for all of these coating purposes (imagine a biofilm jacket), an hydrophobic biofilm coating could be applied in fields ranging from anti-fouling coatings on ships, peers and buoys, to coatings used to protect catheters and protheses from pathogenic bacteria. Moreover, with introducing our kill switch and our chaplin coating without living bacteria, a even broader range of applications can be taken into consideration as certain ethicial and safety issues are dealth with.<br />
<br />
<br><br />
The main advantage of a biofilm coating is that it is very cheap, applying it requires no technical pinnacles and it is more environmentally friendly than certain chemical coatings. Next to that, biofilms can grow on a wide variety of surfaces: They are found on your teeth, in catheters, in plumbing, in water cleaning installations (beneficial), in bioreactors and, if you're one of those students, on your bathroom floor (blech).<br />
<br />
<br><br />
So far there have been coatings with biological substances, but bacteria where only used to produce the coating material. In our project the bacteria will form a biofilm on the desired surface, which will then function as a coating. Our bacteria therefore execute the coating process themselves, which could save a lot of effort. In the case of using chaplins as a building blocks for a hydrophobic coating, the biofilm is also used to orient and anchor the proteins in the right way. As chaplins are amphipathic, the orientation in their pure form is determined by the properties of the surface they coat as well: Using a biofilm to structure them the right way is an easy and smart solution that helps us to get around some problems of using the chaplins in their pure form as a coating.<br />
<br />
<br><br />
<br />
===Medical coatings===<br />
Because of their surface modifying abilities, hydrophobic biofilms of none-pathogenic bacteria may be used to prevent pathogenic biofilms from adhering to prothesis or catheters. Keeping in mind that growing a biofilm, even our ''good'' biofilm coating, in a catheter or on prothesis can give serious medical problems like shock or inflammation. Therefore extensive research would need to be done before our this would be applicable. ''Bacillus subtilis'' is not pathogenic but if needed another bacterium could be used to form the hydrophobic biofilm like ''Lactococcus'', a natural inhabitant of our body. The principle via which we form a ''Bacillus'' biofilm coating is applicable to a number of hosts. Next to that, research could be done to perhaps alter the chaplins so the coating could be functionalized for drug delivery, cell adhesion or anti-bacterial activity.<br />
<br><br />
<br />
===Concrete protection===<br />
As the [https://2010.igem.org/Team:Newcastle Newcastle] team enters this years’ competition with a ''B. subtilis'' machine that fixes cracks in concrete, preventing corrosion and water- or frost-induced damage to buildings, roads or monuments; we have a great addition to their project (or the other way around). Hydrophobic coatings are used to protect concrete structures from corrosion damage, especially when reinforced with metal bars. Our ''B. subtilis'' biofilm would be able to do the same thing by filling up cracks and reducing water inflow by chaplin production: thus preventing damage by water or ice.<br />
<br />
<br><br />
A combination of both projects would make for great bacterial machinery: As the crack-filling bacteria repel the water and ice that is damaging concrete structures further damage is prevented and repair is accelerated.</div>Ekkershttp://2010.igem.org/Team:Groningen/ApplicationsTeam:Groningen/Applications2010-10-28T00:15:59Z<p>Ekkers: /* Anti-fouling coatings */</p>
<hr />
<div>__NOTOC__<br />
==Applications==<br />
A biofilm coating can be used for a great number of applications. We developed our hydrophobic coating keeping in mind that future development of our project could focus on adding other properties to a biofilm coating as well, creating functional coatings with different purposes. Utilizing the qualities of different biofilm forming bacteria or micro organisms and combining these coating properties with the ever growing stack of biobricks within the database could give rise to a variety of engineered biofilm machines, capable of transforming surfaces under many conditions.<br />
<br />
===Hydrophobic coatings===<br />
The potential benefits of hydrophobic coatings can be put to use in a wide variety of application areas; hydrophobic clothes, anti-fouling coatings, anti-corrosion coatings, hydrophobic paint and medical coatings for drug delivery or against biofilm formation. For these applications a lot of hydrophobic coatings have been developed. Not all of these coatings have been succesfully used outside the lab, so the search for a cheap, multi-functional hydrophobic coating is still ongoing.<br />
<br />
<br><br />
<br />
===Anti-fouling coatings===<br />
Surface hydrophobicity could also be beneficial in the antifouling industry, for instance when applied on ships. When marine micro-organisms like algae or pocks adhere to the hull of ships, they form a layer which greatly increases drag in the water. This results in higher fuel costs and increased erosion. To prevent organisms to adhere to the hull of ships, chemical antifouling paints, which often contain copper and tin, are used. 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 tons of organotin and 30 tons of copper end up in the environment every year. With our biological coating this impact on the environment could be greatly reduced.<br />
<br />
<br><br />
<br />
'''Must be''' <br />
Anticorrosive <br />
Antifouling <br />
Environmentally acceptable <br />
Economically viable <br />
Long life <br />
A target for non-specific species<br />
Compatible with underlying system<br />
Resistant to abrasion/biodegradation/erosion<br />
Capable of protecting regardless of operational profile<br />
Smooth<br />
<br />
<br><br />
<br />
'''Must not be:'''<br />
Toxic to the environment<br />
Persistent in the environment<br />
Expensive<br />
Chemically unstable<br />
A target for non-specific species<br />
<br />
<br><br />
<br />
Organotin pollution in China: an overview of the current state and potential health risk - Cao et al.<br />
<br />
===Biofilm coatings===<br />
<br />
Although a biofilm is not ideal for all of these coating purposes (imagine a biofilm jacket), an hydrophobic biofilm coating could be applied in fields ranging from anti-fouling coatings on ships, peers and buoys, to coatings used to protect catheters and protheses from pathogenic bacteria. Moreover, with introducing our kill switch and our chaplin coating without living bacteria, a even broader range of applications can be taken into consideration as certain ethicial and safety issues are dealth with.<br />
<br />
<br><br />
The main advantage of a biofilm coating is that it is very cheap, applying it requires no technical pinnacles and it is more environmentally friendly than certain chemical coatings. Next to that, biofilms can grow on a wide variety of surfaces: They are found on your teeth, in catheters, in plumbing, in water cleaning installations (beneficial), in bioreactors and, if you're one of those students, on your bathroom floor (blech).<br />
<br />
<br><br />
So far there have been coatings with biological substances, but bacteria where only used to produce the coating material. In our project the bacteria will form a biofilm on the desired surface, which will then function as a coating. Our bacteria therefore execute the coating process themselves, which could save a lot of effort. In the case of using chaplins as a building blocks for a hydrophobic coating, the biofilm is also used to orient and anchor the proteins in the right way. As chaplins are amphipathic, the orientation in their pure form is determined by the properties of the surface they coat as well: Using a biofilm to structure them the right way is an easy and smart solution that helps us to get around some problems of using the chaplins in their pure form as a coating.<br />
<br />
<br><br />
<br />
===Medical coatings===<br />
Because of their surface modifying abilities, hydrophobic biofilms of none-pathogenic bacteria may be used to prevent pathogenic biofilms from adhering to prothesis or catheters. Keeping in mind that growing a biofilm, even our ''good'' biofilm coating, in a catheter or on prothesis can give serious medical problems like shock or inflammation. Therefore extensive research would need to be done before our this would be applicable. ''Bacillus subtilis'' is not pathogenic but if needed another bacterium could be used to form the hydrophobic biofilm like ''Lactococcus'', a natural inhabitant of our body. The principle via which we form a ''Bacillus'' biofilm coating is applicable to a number of hosts. Next to that, research could be done to perhaps alter the chaplins so the coating could be functionalized for drug delivery, cell adhesion or anti-bacterial activity.<br />
<br><br />
<br />
===Concrete protection===<br />
As the [https://2010.igem.org/Team:Newcastle Newcastle] team enters this years’ competition with a ''B. subtilis'' machine that fixes cracks in concrete, preventing corrosion and water- or frost-induced damage to buildings, roads or monuments; we have a great addition to their project (or the other way around). Hydrophobic coatings are used to protect concrete structures from corrosion damage, especially when reinforced with metal bars. Our ''B. subtilis'' biofilm would be able to do the same thing by filling up cracks and reducing water inflow by chaplin production: thus preventing damage by water or ice.<br />
<br />
<br><br />
A combination of both projects would make for great bacterial machinery: As the crack-filling bacteria repel the water and ice that is damaging concrete structures further damage is prevented and repair is accelerated.</div>Ekkershttp://2010.igem.org/Team:Groningen/ApplicationsTeam:Groningen/Applications2010-10-28T00:13:52Z<p>Ekkers: /* Anti-fouling coatings */</p>
<hr />
<div>__NOTOC__<br />
==Applications==<br />
A biofilm coating can be used for a great number of applications. We developed our hydrophobic coating keeping in mind that future development of our project could focus on adding other properties to a biofilm coating as well, creating functional coatings with different purposes. Utilizing the qualities of different biofilm forming bacteria or micro organisms and combining these coating properties with the ever growing stack of biobricks within the database could give rise to a variety of engineered biofilm machines, capable of transforming surfaces under many conditions.<br />
<br />
===Hydrophobic coatings===<br />
The potential benefits of hydrophobic coatings can be put to use in a wide variety of application areas; hydrophobic clothes, anti-fouling coatings, anti-corrosion coatings, hydrophobic paint and medical coatings for drug delivery or against biofilm formation. For these applications a lot of hydrophobic coatings have been developed. Not all of these coatings have been succesfully used outside the lab, so the search for a cheap, multi-functional hydrophobic coating is still ongoing.<br />
<br />
<br><br />
<br />
===Anti-fouling coatings===<br />
Surface hydrophobicity could also be beneficial in the antifouling industry, for instance when applied on ships. When marine micro-organisms like algae or pocks adhere to the hull of ships, they form a layer which greatly increases drag in the water. This results in higher fuel costs and increased erosion. To prevent organisms to adhere to the hull of ships, chemical antifouling paints, which often contain copper and tin, are used. 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 tons of organotin and 30 tons of copper end up in the environment every year. With our biological coating this impact on the environment could be greatly reduced.<br />
''Must be'' <br />
Anticorrosive <br />
Antifouling <br />
Environmentally acceptable <br />
Economically viable <br />
Long life <br />
A target for non-specific species<br />
Compatible with underlying system<br />
Resistant to abrasion/biodegradation/erosion<br />
Capable of protecting regardless of operational profile<br />
Smooth<br />
<br><br />
''Must not be:''<br />
Toxic to the environment<br />
Persistent in the environment<br />
Expensive<br />
Chemically unstable<br />
A target for non-specific species<br />
<br />
<br><br />
<br />
Organotin pollution in China: an overview of the current state and potential health risk - Cao et al.<br />
<br />
===Biofilm coatings===<br />
<br />
Although a biofilm is not ideal for all of these coating purposes (imagine a biofilm jacket), an hydrophobic biofilm coating could be applied in fields ranging from anti-fouling coatings on ships, peers and buoys, to coatings used to protect catheters and protheses from pathogenic bacteria. Moreover, with introducing our kill switch and our chaplin coating without living bacteria, a even broader range of applications can be taken into consideration as certain ethicial and safety issues are dealth with.<br />
<br />
<br><br />
The main advantage of a biofilm coating is that it is very cheap, applying it requires no technical pinnacles and it is more environmentally friendly than certain chemical coatings. Next to that, biofilms can grow on a wide variety of surfaces: They are found on your teeth, in catheters, in plumbing, in water cleaning installations (beneficial), in bioreactors and, if you're one of those students, on your bathroom floor (blech).<br />
<br />
<br><br />
So far there have been coatings with biological substances, but bacteria where only used to produce the coating material. In our project the bacteria will form a biofilm on the desired surface, which will then function as a coating. Our bacteria therefore execute the coating process themselves, which could save a lot of effort. In the case of using chaplins as a building blocks for a hydrophobic coating, the biofilm is also used to orient and anchor the proteins in the right way. As chaplins are amphipathic, the orientation in their pure form is determined by the properties of the surface they coat as well: Using a biofilm to structure them the right way is an easy and smart solution that helps us to get around some problems of using the chaplins in their pure form as a coating.<br />
<br />
<br><br />
<br />
===Medical coatings===<br />
Because of their surface modifying abilities, hydrophobic biofilms of none-pathogenic bacteria may be used to prevent pathogenic biofilms from adhering to prothesis or catheters. Keeping in mind that growing a biofilm, even our ''good'' biofilm coating, in a catheter or on prothesis can give serious medical problems like shock or inflammation. Therefore extensive research would need to be done before our this would be applicable. ''Bacillus subtilis'' is not pathogenic but if needed another bacterium could be used to form the hydrophobic biofilm like ''Lactococcus'', a natural inhabitant of our body. The principle via which we form a ''Bacillus'' biofilm coating is applicable to a number of hosts. Next to that, research could be done to perhaps alter the chaplins so the coating could be functionalized for drug delivery, cell adhesion or anti-bacterial activity.<br />
<br><br />
<br />
===Concrete protection===<br />
As the [https://2010.igem.org/Team:Newcastle Newcastle] team enters this years’ competition with a ''B. subtilis'' machine that fixes cracks in concrete, preventing corrosion and water- or frost-induced damage to buildings, roads or monuments; we have a great addition to their project (or the other way around). Hydrophobic coatings are used to protect concrete structures from corrosion damage, especially when reinforced with metal bars. Our ''B. subtilis'' biofilm would be able to do the same thing by filling up cracks and reducing water inflow by chaplin production: thus preventing damage by water or ice.<br />
<br />
<br><br />
A combination of both projects would make for great bacterial machinery: As the crack-filling bacteria repel the water and ice that is damaging concrete structures further damage is prevented and repair is accelerated.</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T23:51:16Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
''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)<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
[[Image:agar TY corn starch.jpg|right|300px]]<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''<br />
<br />
<br><br />
<br />
==References==<br />
<br />
1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms<br />
<br />
2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
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</div>Ekkershttp://2010.igem.org/File:Agar_TY_corn_starch.jpgFile:Agar TY corn starch.jpg2010-10-27T23:49:38Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T23:49:03Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
''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)<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
[[Image:agar TY corn starch.jpg|right]]<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''<br />
<br />
<br><br />
<br />
==References==<br />
<br />
1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms<br />
<br />
2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
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</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T23:34:12Z<p>Ekkers: /* References */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
''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)<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''<br />
<br />
<br><br />
<br />
==References==<br />
<br />
1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms<br />
<br />
2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
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</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T23:29:29Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
''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)<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''<br />
<br />
<br><br />
<br />
==References==<br />
<br />
1. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
2. 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</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T23:25:14Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''<br />
<br />
<br><br />
<br />
==References==<br />
<br />
1. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
2. 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</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T23:22:19Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''<br />
<br />
<br><br />
<br />
----<br />
<br />
==References==<br />
<br />
1. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
2. 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</div>Ekkershttp://2010.igem.org/File:THT_Cinducedgr.jpgFile:THT Cinducedgr.jpg2010-10-27T23:01:12Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-27T22:59:11Z<p>Ekkers: /* Subtilin induced expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
<br><br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Groningen-ODvsFluor-GFP.png|right|300px]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
[[Image:THT Cinducedgr.png|right|200px]]<br />
[[Image:THT EH gr.png|right|200px]]<br />
[[Image:THT Einducedgr.png|right|200px]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Ekkershttp://2010.igem.org/File:THT_E_gr.pngFile:THT E gr.png2010-10-27T22:56:01Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-27T22:44:14Z<p>Ekkers: /* Subtilin induced expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
<br><br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Groningen-ODvsFluor-GFP.png|right|300px]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
[[Image:THT C gr.png|right|200px]]<br />
[[Image:THT EH gr.png|right|200px]]<br />
[[Image:THT E1 gr.png|right|200px]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Ekkershttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:22:29Z<p>Ekkers: /* Self assembling hydrophobic biofilm */</p>
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A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
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<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
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<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
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===Self assembling hydrophobic biofilm===<br />
'''We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].'''<br />
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This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
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<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
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<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
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=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
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</html></div>Ekkershttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:21:59Z<p>Ekkers: /* Self assembling hydrophobic biofilm */</p>
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</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
<br />
<br />
<div margin-left: 10px"> <br />
</html><br />
===Self assembling hydrophobic biofilm===<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
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<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
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<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
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<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
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</html></div>Ekkershttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:21:27Z<p>Ekkers: /* Self assembling hydrophobic biofilm */</p>
<hr />
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<img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /><br />
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A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
<br />
<br />
<div margin-left: 10px"> <br />
</html><br />
===Self assembling hydrophobic biofilm===<br />
'''We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].''' <br />
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<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
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<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<br />
<br />
<html><br />
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</html></div>Ekkershttp://2010.igem.org/File:THT_EH_gr.pngFile:THT EH gr.png2010-10-27T22:07:31Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/File:THT_C_gr.jpgFile:THT C gr.jpg2010-10-27T21:58:51Z<p>Ekkers: </p>
<hr />
<div></div>Ekkershttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-27T21:45:52Z<p>Ekkers: /* Subtilin induced expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
<br><br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Groningen-ODvsFluor-GFP.png|right|300px]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and TCA precipitated supernatant with TFA to purify chaplin proteins. TFA treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] This turned out to be a more successful method.<br />
[[Image:THT C gr.bmp|right|200px]]<br />
[[Image:THT EH gr.bmp|right|200px]]<br />
[[Image:THT E1 gr.bmp|right|200px]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Ekkershttp://2010.igem.org/Team:Groningen/ProjectTeam:Groningen/Project2010-10-27T21:09:04Z<p>Ekkers: /* Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
</html><br />
<br />
== Self assembling hydrophobic biofilm ==<br />
[[Image:chaplin cell.jpg|right]]<br />
Surface hydrophobicity is a very useful property and is used in many applications ranging from raincoats, antifouling coatings, anti-bacterial and anti-fungal coatings and applications in the field of biomedical sciences to protection of highly sensitive sensor equipment. Hydrophobicity keeps a surface water free and thereby clean and dry, this prevents micro-organisms from fouling surfaces and corrosion from forming. Most hydrophobic coatings used today are either costly or contain harmful chemicals. So why not create an organism that does the work for you. <br />
<br />
The idea is to engineer a bacterium that once applied on a surface, starts forming a [https://2010.igem.org/Team:Groningen#/biofilmfast rigid biofilm]. Completion of the biofilm will trigger the [https://2010.igem.org/Team:Groningen#/hydrophobins expression of hydrophobic proteins] by the biofilm forming bacterium. These hydrophobic proteins will be incorporated in the rigid biofilm, causing strong hydrophobic surface activity. After the hydrophobic biofilm production, a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] would be activated to kill of all bacteria. The result of these processes will be a surface that is coated by a rigid biofilm with embedded hydrophobic proteins, leaving the coated surface extremely hydrophobic.<br />
<br />
Producing a hydrophobic biocoating that is self assembling, would have a lot of advantages.<br />
Firstly it is relatively cheap to apply bacteria to surfaces. And since the coating process will be done by the bacteria themselves, there is no high-tech treatment involved and there are no expensive chemicals necessary to attain the hydrophobicity.<br />
Secondly, because these hydrophobins are proteins, they are in contrast to many chemical hydrophobins non-toxic to the environment. Applications of this hydrophobic biofilm could range from antifouling coatings on ships, antifungal coatings and corrosion and water protecting coatings.</div>Ekkershttp://2010.igem.org/Team:Groningen/ProjectTeam:Groningen/Project2010-10-27T21:08:27Z<p>Ekkers: /* Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
</html><br />
<br />
== Self assembling hydrophobic biofilm ==<br />
[[Image:chaplin cell.jpg|right]]<br />
Surface hydrophobicity is a very useful property and is used in many applications ranging from raincoats, antifouling coatings, anti-bacterial and anti-fungal coatings and applications in the field of biomedical sciences to protection of highly sensitive sensor equipment. Hydrophobicity keeps a surface water free and thereby clean and dry, this prevents micro-organisms from fouling surfaces and corrosion from forming. Most hydrophobic coatings used today are either costly or contain harmful chemicals. So why not create an organism that does the work for you. <br />
<br />
The idea is to engineer a bacterium that once applied on a surface, starts forming a [https://2010.igem.org/Team:Groningen#/biofilmfast growing rigid biofilm]. Completion of the biofilm will trigger the [https://2010.igem.org/Team:Groningen#/hydrophobins expression of hydrophobic proteins] by the biofilm forming bacterium. These hydrophobic proteins will be incorporated in the rigid biofilm, causing strong hydrophobic surface activity. After the hydrophobic biofilm production, a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] would be activated to kill of all bacteria. The result of these processes will be a surface that is coated by a rigid biofilm with embedded hydrophobic proteins, leaving the coated surface extremely hydrophobic.<br />
<br />
Producing a hydrophobic biocoating that is self assembling, would have a lot of advantages.<br />
Firstly it is relatively cheap to apply bacteria to surfaces. And since the coating process will be done by the bacteria themselves, there is no high-tech treatment involved and there are no expensive chemicals necessary to attain the hydrophobicity.<br />
Secondly, because these hydrophobins are proteins, they are in contrast to many chemical hydrophobins non-toxic to the environment. Applications of this hydrophobic biofilm could range from antifouling coatings on ships, antifungal coatings and corrosion and water protecting coatings.</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T20:57:15Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T20:56:21Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/expression expression page] or the[https://2010.igem.org/Team:Groningen#/modeling modeling page]. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T20:51:45Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/modeling modeling page]. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
<br />
<br><br />
<br />
''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.''</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T20:50:31Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/modeling modeling page]. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
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.</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T20:49:50Z<p>Ekkers: /* Biofilm */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/modeling modeling page]. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
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. <br />
<br />
<br><br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
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.<br />
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.</div>Ekkershttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-27T20:48:53Z<p>Ekkers: /* References */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
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 [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://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.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
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. <br />
<br />
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. <br />
<br />
''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.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
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[[article Akos]].<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
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. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''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). [https://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 [https://2010.igem.org/Team:Groningen#/modeling modeling page]. <br />
<br />
<br><br />
<br />
'''Coating surfaces'''<br />
<br />
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 <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://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.<br />
<br />
<br><br />
<br />
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. <br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
<br />
<br><br />
<br />
''Biofilm paste''<br />
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 [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
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.<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
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.</div>Ekkers