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{{Imperial/StartPage}}
{{Imperial/StartPage2}} __NOTOC__
<br>
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&nbsp; <font size=6px color=#E5EBFF><b>Why ''B. subtilis''?</b></font>
<br><br>
{{Imperial/Box2||This page offers a brief overview of how ''B. subtilis'' meets our main project specifications - to a much higher degree than ''E. coli''! You will find information on the main mechanisms behind our biofabricator and some very interesting biology too. If you would like even ''more'' in-depth information, please click on ''Details'' under each section.
|}}


Authors: Wiki team
{{Imperial/Box1|Light Sensing|We need a trigger - a stimulus that our biofabricators can detect and respond to quickly - such that we can control the synthesis of our biomaterial in a set pattern in 3D.


Editors: Wiki team
Light is the most obvious candidate, as holography allows us to generate complex patterns with well defined edges in 3D. After examining a number of light sensing pathways, we decided to utilise a native pathway involving YtvA, which is a protein used by ''B. subtilis'' to detect blue light. YtvA triggers a cascade of interactions, but some way down the chain, a molecule called sigma B (σ<sub>B</sub>) is produced. This, in turn, boosts the synthesis of YtvA.


= Chassis 1 =
We plan to over-express YtvA and use σ<sub>B</sub> as a promoter for genes which stop movement and produce biomaterial. Therefore, when the bacteria detect blue light, those genes will turn on, the bacteria will stop and biomaterial synthesis will begin. <br><br>[[IGEM:IMPERIAL/2008/Prototype/Light| '''>>> Details >>>''']]|[[Image:Imperial_2008_Holgram_Art.jpg |200px | 3D blue holographic image by sculptor Eileen Borgeson[http://www.eileenborgeson.com/default.htm]]}}


This page offers a brief overview of how ''B. subtilis'' meets our main project specifications - to a much higher degree than ''E. coli''! You'll find information on the main mechanisms behind our proposed system, and some very interesting biology too. If you would like even ''more'' depth than the outlines below, you are welcome to visit our OpenWetWare pages on those topics - but don't get to engrossed and forget to come back!


==Motility==
{{Imperial/Box1|Motility|To achieve accurate distribution of our biofabricators, we must exert fine control over their motility. Bacteria's primary method of getting about is via flagellar locomotion. A protein ring on the cell membrane is attached to the flagellum and rotates during locomotion, acting like a propeller to push the cell through its environment.
[[Image:B_subtilis_Clutch_Mechanism.png|400px|thumb|A schematic of the ''B. subtilis'' rotary flagellar motor is shown. Motile cells are powered by interactions of the FliG protein with the MotA/B complex (which generates torque). The protein EpsE acts as a molecular clutch to disengage the rotary flagellar motor, leaving the flagellum intact but unpowered. This shuts down motility and facilitates biofilm formation. Fluorescence microscopy photos of ''B. subtilis'' show bacterial membranes in red and flagella in green. FliM and FliF are motor proteins [http://www.sciencemag.org/cgi/reprint/320/5883/1599.pdf]]]


To achieve accurate distribution of our biomaterial microfactories (an affectionate term for our bacteria) we need to be able to exert fine control over their motility. ''B. subtilis''' prime method of getting about is flagellar locomotion; a ring of protein in the cell membrane rotates and is attached to a flagella that extrudes, acting like a long corkscrew propeller to push the cell through its environment.
The precise mechanism of how this works in ''B. subtilis'' has recently been elucidated. The flagella can be detached from the rotor by expression of a clutch molecule that interacts with the flagella and distorts it, so it is disengaged from the rotor protein. Control over the expression of this clutch should allow us to control the bacteria very quickly. When we want the bacteria to stop, we trigger expression of the clutch, which halts movement.


Where ''B. subtilis'' differs from other bacteria is in our knowledge of its mechanism for this movement. A recent paper [LINK] described a clutch mechanism involved in the process; the flagella can be detached from the rotor by expression of a molecule that interacts with the flagella and distorts it so it is disengaged from the rotor protein.
To draw a parallel with a car, currently available synthetic methods of stopping bacteria are akin to destroying the engine. Our method is analogous to putting the car into neutral - disengaging the engine from the driveshaft. It is an elegant solution that offers us quick control and also the opportunity for quick reversal (putting the car back into "drive").
<br><br>
[[IGEM:IMPERIAL/2008/Prototype/Motility | '''>>> Details >>>''']]
|[[Image:B_subtilis_Clutch_Mechanism.png|center|400px|Motile ''B. subtilis'' cells are powered by interactions between protein complexes, generating torque for locomotion. The protein EpsE acts as a molecular clutch to disengage the flagellar motor, leaving the flagellum intact but unpowered. This quickly halts locomotion[http://www.sciencemag.org/cgi/reprint/320/5883/1599.pdf]]]}}


Control over the expression of this protein should allow us very quick control of the bacteria - when we want it to stop we trigger expression of the clutch molecule which halts movement.


To draw a parallel with a car, current synthetic methods of stopping bacteria are akin to destroying the engine (which can take time!) in order to stop it powering the vehicle where the method we hope to take advantage of would be like putting the car into neutral - disengaging the engine from the driveshaft. It's an elegant solution that offers us quick control and also the opportunity for quick reversal (putting the car back into "drive").


More: http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Motility
{{Imperial/Box1|Biomaterial Synthesis|After our bacteria are positioned correctly, they need to express a biomaterial. ''B. subtilis'' is Gram-positive, meaning it has only a single membrane as opposed to a double membrane in Gram-negative bacteria like ''E. coli''. This means that the expression of a biomaterial is a lot more tractable; biomaterial can be produced and secreted more efficiently. With a double membrane, material may accumulate inside the cell and the efficiency of biomaterial production can be significantly lower.
<br clear="all">


==Light Sensing==
Another important aspect of our biomaterial specifications is what we want to secrete. We did a lot of research on this and decided to express elastin peptides and EAK16-II. Both are small peptides and their molecular structures favour their self-assembly outside the ''B. subtilis'' cells to form 3D bio-scaffolds.
<br><br>
3D bio-scaffolds are very useful for tissue culture and regenerative medicine, as they offer a suitable 3D enviroment for implanted cells to grow and proliferate. A good analogy would be scaffolding used in the construction industry. Our blue-sky aim is to construct a genetically-engineered machine that can fabricate bio-scaffolds with precise 3D shapes, directed by 3D holography.
<br><br>
[[IGEM:IMPERIAL/2008/Prototype/Biomaterials | '''>>> Details >>>''']]|[[Image:Imperial_2008_Scaffold.jpg|right|300px|3D Scaffold]]}}
<br clear="all"><hr>
{{Imperial/Box1||So ''B. subtilis'' fulfils our main specifications perfectly, and can be made to meet our minor specifications with relatively ease. On top of that, it does have other benefits, along with some challenges. These are listed on the next page, together with an overview of our development of ''B. subtilis'' as a chassis.
<br><br>
[[IGEM:IMPERIAL/2008/New/Chassis_2 | '''>>> Benefits ''vs'' Challenges >>>''']]}}


We have chosen light stimulus as our input for the Bioprinter. Light allows us to generate complex patterns with well defined edges, while gradients in wavelength and intensity will allow us to build up varying concentrations of biomaterial.
{{Imperial/EndPage|Project|Chassis_2}}
 
Photoreceptors which were considered in our design include rhodopsins, phytochromes, LOV and BLUF. Of these, B.Subtilis contains the blue light sensing protein YtvA which is related to the plant blue-light receptors phototropins. YtvA is a positive activator of the general stress transcription factor σB. Blue and not red light activates YtvA and thus induces σB activity. We could use σB which is expressed upon blue light illumination to activate the production of EpsE and hence control bacteria motility. However, there are other stressors which activate σB, hence we aim to make blue light the main activator of σB by controlling growth conditions.
 
==Biomaterials==
 
http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Biomaterials
 
 
<hr>
 
 
The next page details some more pros and cons about working with ''subtilis'', as well as an overview of our development of it as a chassis... [[IGEM:IMPERIAL/2008/New/Chassis_2 | '''> The Second Chassis Page >''']]
 
{{Imperial/EndPage}}

Latest revision as of 07:17, 9 October 2008

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       <a href="http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Chassis_2">B. subtilis: Benefits vs Challenges</a>
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  Why B. subtilis?

This page offers a brief overview of how B. subtilis meets our main project specifications - to a much higher degree than E. coli! You will find information on the main mechanisms behind our biofabricator and some very interesting biology too. If you would like even more in-depth information, please click on Details under each section.


Light Sensing

We need a trigger - a stimulus that our biofabricators can detect and respond to quickly - such that we can control the synthesis of our biomaterial in a set pattern in 3D.

Light is the most obvious candidate, as holography allows us to generate complex patterns with well defined edges in 3D. After examining a number of light sensing pathways, we decided to utilise a native pathway involving YtvA, which is a protein used by B. subtilis to detect blue light. YtvA triggers a cascade of interactions, but some way down the chain, a molecule called sigma B (σB) is produced. This, in turn, boosts the synthesis of YtvA.

We plan to over-express YtvA and use σB as a promoter for genes which stop movement and produce biomaterial. Therefore, when the bacteria detect blue light, those genes will turn on, the bacteria will stop and biomaterial synthesis will begin.

>>> Details >>>

3D blue holographic image by sculptor Eileen Borgeson[http://www.eileenborgeson.com/default.htm



Motility

To achieve accurate distribution of our biofabricators, we must exert fine control over their motility. Bacteria's primary method of getting about is via flagellar locomotion. A protein ring on the cell membrane is attached to the flagellum and rotates during locomotion, acting like a propeller to push the cell through its environment.

The precise mechanism of how this works in B. subtilis has recently been elucidated. The flagella can be detached from the rotor by expression of a clutch molecule that interacts with the flagella and distorts it, so it is disengaged from the rotor protein. Control over the expression of this clutch should allow us to control the bacteria very quickly. When we want the bacteria to stop, we trigger expression of the clutch, which halts movement.

To draw a parallel with a car, currently available synthetic methods of stopping bacteria are akin to destroying the engine. Our method is analogous to putting the car into neutral - disengaging the engine from the driveshaft. It is an elegant solution that offers us quick control and also the opportunity for quick reversal (putting the car back into "drive").

>>> Details >>>

Motile B. subtilis cells are powered by interactions between protein complexes, generating torque for locomotion. The protein EpsE acts as a molecular clutch to disengage the flagellar motor, leaving the flagellum intact but unpowered. This quickly halts locomotion[1]
Motile B. subtilis cells are powered by interactions between protein complexes, generating torque for locomotion. The protein EpsE acts as a molecular clutch to disengage the flagellar motor, leaving the flagellum intact but unpowered. This quickly halts locomotion[1]


Biomaterial Synthesis

After our bacteria are positioned correctly, they need to express a biomaterial. B. subtilis is Gram-positive, meaning it has only a single membrane as opposed to a double membrane in Gram-negative bacteria like E. coli. This means that the expression of a biomaterial is a lot more tractable; biomaterial can be produced and secreted more efficiently. With a double membrane, material may accumulate inside the cell and the efficiency of biomaterial production can be significantly lower.

Another important aspect of our biomaterial specifications is what we want to secrete. We did a lot of research on this and decided to express elastin peptides and EAK16-II. Both are small peptides and their molecular structures favour their self-assembly outside the B. subtilis cells to form 3D bio-scaffolds.

3D bio-scaffolds are very useful for tissue culture and regenerative medicine, as they offer a suitable 3D enviroment for implanted cells to grow and proliferate. A good analogy would be scaffolding used in the construction industry. Our blue-sky aim is to construct a genetically-engineered machine that can fabricate bio-scaffolds with precise 3D shapes, directed by 3D holography.

>>> Details >>>

3D Scaffold
3D Scaffold


So B. subtilis fulfils our main specifications perfectly, and can be made to meet our minor specifications with relatively ease. On top of that, it does have other benefits, along with some challenges. These are listed on the next page, together with an overview of our development of B. subtilis as a chassis.

>>> Benefits vs Challenges >>>



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