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''B.subtilis'' as a chassis made its first appearance last year and has many advantages for use at iGEM including but not limited to; a single membrane allowing for easier secretion, its high motility and a large amount of data in the literature. There are however very few parts for ''B.subtilis'' meaning any team that works in this chassis will need to develop many of the parts for devices and systems from scratch. | ''B.subtilis'' as a chassis made its first appearance last year and has many advantages for use at iGEM including but not limited to; a single membrane allowing for easier secretion, its high motility and a large amount of data in the literature. There are however very few parts for ''B.subtilis'' meaning any team that works in this chassis will need to develop many of the parts for devices and systems from scratch. | ||
For our project, ''B.subtilis'' is vital. ''E.coli'' cannot secrete proteins to any high degree and modifications to it to allow this have not always proven particularly succesful. ''B.subtilis'' has however been used for this purpose for many decades and though there are some technical issues associated with this, it has preformed the job admirably. ''B.subtilis'' is also highly motile and posses a unique (at present) system of switching off its motility in the form of a molecular clutch, EpsE. | For our project, ''B.subtilis'' is vital. ''E.coli'' cannot secrete proteins to any high degree and modifications to it to allow this have not always proven particularly succesful. ''B.subtilis'' has however been used for this purpose for many decades and though there are some technical issues associated with this, it has preformed the job admirably. ''B.subtilis'' is also highly motile and posses a unique (at present) system of switching off its motility in the form of a molecular clutch, EpsE (see Motility below). | ||
= Light Sensing = | = Light Sensing = | ||
Revision as of 05:16, 6 August 2008
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| <html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> | Home | The Project | B.subtilis Chassis | Wet Lab | Dry Lab | Notebook |
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B. subtilis
B.subtilis as a chassis made its first appearance last year and has many advantages for use at iGEM including but not limited to; a single membrane allowing for easier secretion, its high motility and a large amount of data in the literature. There are however very few parts for B.subtilis meaning any team that works in this chassis will need to develop many of the parts for devices and systems from scratch.
For our project, B.subtilis is vital. E.coli cannot secrete proteins to any high degree and modifications to it to allow this have not always proven particularly succesful. B.subtilis has however been used for this purpose for many decades and though there are some technical issues associated with this, it has preformed the job admirably. B.subtilis is also highly motile and posses a unique (at present) system of switching off its motility in the form of a molecular clutch, EpsE (see Motility below).
Light Sensing
If one begins with a lawn of bacteria on a plate, how could production and secretion of biomaterial be achieved in a fixed pattern? Various stimuli include electromagnetic field, chemical and light stimulus. Of these, light allows us to produce materials in a very complex pattern with well defined edges. And if need be, allow gradients of production by varying wavelength and intensity of illumination. Repeating this multiple times for different layers could, therefore, allow us to build up a construction (not unlike a 3D printer [2]) of our material.
The most commonly studied photoreceptors include: rhodopsin (Archaea), phytochromes (cyanobacteria), photoactive yellow proteins, LOV and BLUF. Most of these function through the binding of a photon which causes a conformation change, often in the form of photoisomerisation. Others exhibit oxidation or H-bond strenghtening upon photon binding. Of these, the protein YtvA which contains an LOV domain exists naturally in B.Subtilis. Using other photoreceptors which are not expressed naturally in B.Subtilis might require the design of a fusion protein which may give rise to increased complexity in our project. We have thus decided to utilise the native protein YtvA in our system to function as a blue light receptor.
YtvA from B. subtilis is a flavo-protein related to the plant blue-light receptors phototropins. It is a positive activator of the general stress transcription factor σB. In B. subtilis, σB controls more than 200 genes. Blue and not red light induces σB activity. We could thus use σB which is expressed upon blue light illumination to activate the production of EpsE and hence control bacteria motility. Possible complications arising from the use of this pathway include the effects of the stress response activated by σB. To understand the pathway in more detail we have looked at the other stress conditions that lead to the activation of σB.
References (light section)
- New Insights into Metabolic Properties of Marine Bacteria Encoding Proteorhodopsins (Would be good if someone could decode what this paper is saying) [3]
- Light powered E.Coli with Proteorhodopsin [4]
- Blue Light Activates the σB -Dependent Stress Response of Bacillus subtilis via YtvA. Marcela A´ vila-Pe´rez, Klaas J. Hellingwerf, and Remco Kort*[5]
- First Evidence for Phototropin-Related Blue-Light Receptors in Prokaryotes. Aba Losi, Eugenia Polverini, Benjamin Quest and Wolfgang Gartner [6]
- Listening to the blue: the time-resolved thermodynamics of the bacterial blue-light receptor YtvA and its isolated LOV domain. Aba Losi, Benjamin Quest and Wolfgang Gärtner [7]
- Photosensing in chemotrophic, non-phototrophic bacteria: let there be light sensing too Michael A. van der Horst, Jason Key, and Klaas J. Hellingwerf [8]
Motility
The Flagellar Clutch
Here is a simple explanation of how the clutch system works: What happens is that the gene SinR upregulates the expression of flagellar genes i.e. MotA/MotB etc. and downregulates expression of biofilm forming genes. If SinR is absent, biofilm takes over and the bacteria loses its motility. It was found that SinR represses the EpsE gene, thus the absence of SinR causes the expression of EpsE [9].
It was later determined that EpsE is responsible for disengaging the clutch in the flagellar motor [10]. This means that instead of turning the motor on or off, bacteria's motor is continuously spinning, but disengaging or engaging the flagella to the motor is regulated by EpsE and thus the EpsE gene. If we can characterise the EpsE gene, we should be able to control whether the bacteria moves or not, irregardless of its orientation, which brings us to the next problem.
2005 Penn State Uni team did a similar project, but they didn't use EpsE/SinR gene expression. Instead, they played around with the actual motor of the bacteria, motB gene which generates the torque required to give flagellum its rotating power [11].
Biomaterials
Erika & Prudence - C&P from rough wiki
Progress so Far
The diagram on the right (click for larger image) shows a theoretical overview of the circuitry we'll be adding to B. subtilis in order to control its movement and expression profile.
The top row shows genes native to B. subtilis that may interfere with our aims. These include the endogenous epsE gene, producing the molecular clutch, and the associated sinR gene which inhibits it. The second row shows synthetic constructs which we will add that are to be constitutively expressed and the third row shows synthetic constructs that we require to be inducible. Genes in green generally upregulate/promote transcription of some target whereas those in red downregulate/repress activity of their targets; lines showing interactions follow the same scheme. SinR, for example, inhibits the operon containing the epsE gene so the sinR gene is red. The gene for product production (or reporters) is coloured yellow.
The general operation of these constructs should fulfil our specifications, as the simple explanations below demonstrate...
- No light present: Inducible promoter is not activated, meaning no transcription of product and no production of synthetic EpsE. The constitutive expression of SinR, alongside the cell's own background expression of same, should prevent production of native EpsE so the bacteria will remain motile.
- Light present: Inducible promoter is activated, meaning product is transcribed along with EpsE. Native EpsE is still repressed by the native and synthetic constitutive SinR, but the synthetic EpsE should engage the clutch and halt the bacteria in a short timeframe. The bacteria should, therefore, stop and start producing product.
- Light removed: Should a bacteria move out of the light, we want it to be able to turn off production and start moving again. When light is absent, the inducible promoter will no longer be active and the bacteria should revert to the "no light" state. The time it takes to do this will be directly linked to the accuracy of deposition, and so we want it to be as fast as possible.
As far as characterising parts goes (defining individual parts) we have made some headway. If we use the natural ytvA pathway, that activates σB; a transcription factor required for some promoters to operate. One of these promoters is pctc so if we use that as our inducible promoter, the natural response to light (which we can amplify by adding constitutive expression of those receptors) will kick the bacteria into the production state.
The ExpMach gene is a placeholder; should the biomaterial expression we are trying to output require any expression machinery, the genes for it will probably go here. They could be constitutively expressed with the ytvA and sinR genes but putting them here gives a time delay between the bacteria stopping and product being excreted which could be useful.
As an addition to this diagram, there will probably exist a positive feedback loop for the constitutive block; SinR represses rok which usually represses comK. ComK upregulates several promoters, so if we use one of those promoters in our constitutive block the sinR gene will indirectly upregulate itself.
Potential Issues
The constructs we have designed are polycistronic, B.subtilis translation of polycistronic genes however is not even. The first gene in a polycistron is often more highly expressed than the second even if they have their own individual Ribosome Binding Sites [1]. This may cause issues for our current circuit design but could be easily remedied by using multiple promoters in each construct or by the arrangement of genes along the polycistron.
Also, the differential codon usage of organisms must be taken into account. Some of our proposed Biomaterials are from other organisms, most notably the elastin exon protein which if from the Human genome. If this protein was expressed in its current form in B.subtilis the amino acid code would be very different to that observed upon expression in a human. As such, the elastin exon sequence will need to be 'translated' into a sequence that will give the same product in B.subtilis as the original would in humans.
Team Divisions
At the moment we're separated into two teams:
- The wetlab team (James, Chris, Tom & Krupa) has so far concentrated on the mammoth task of finding sequences and writing protocols, and will move on to setting up and organising experiments.
- The drylab team (Erika, QQ, Prudence, Clinton & Yanis) is working out a strategy for modelling of behaviour of our bacteria on Matlab, and will be using data from a recorded microscope analysis of subtilis motility to help define parameters. More detail can be found on the Wet Lab and Dry Lab pages.
References
When the page is completed, the references from ALL the sections should be collated here
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