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This diagram shows our critical pathway; the cloning steps we will undertake to produce our final constructs (and on the way, produce some valuable test constructs). We aim to re-use as many parts as possible to reduce the number of steps, but because of planned redundancy we often have multiple variants for steps. So for each addition of a promoter, there are actually ''four'' cloning procedures; one for each combination of the two promoters and RBSs we have ordered. | This diagram shows our critical pathway; the cloning steps we will undertake to produce our final constructs (and on the way, produce some valuable test constructs). We aim to re-use as many parts as possible to reduce the number of steps, but because of planned redundancy we often have multiple variants for steps. So for each addition of a promoter, there are actually ''four'' cloning procedures; one for each combination of the two promoters and RBSs we have ordered. | ||
In total, we believe there are | In total, we believe there are 101 cloning steps. | ||
Without the sequences we ordered from Geneart, we can make a start by using the parts acquired from the Registry. These experiments are thus termed phase 0. Phase 1 requires some of our [[#Geneart | Geneart sequences]] - most notably the promoters/RBSs - and is focused on characterisation of the constitutive promoters. Phases 2 and 3 involve construction of test constructs to characterise our inducible promoters (which will lead our epsE and biomaterial genes) and phase 4 looks at light-inducible promoters. | Without the sequences we ordered from Geneart, we can make a start by using the parts acquired from the Registry. These experiments are thus termed phase 0. Phase 1 requires some of our [[#Geneart | Geneart sequences]] - most notably the promoters/RBSs - and is focused on characterisation of the constitutive promoters. Phases 2 and 3 involve construction of test constructs to characterise our inducible promoters (which will lead our epsE and biomaterial genes) and phase 4 looks at light-inducible promoters. | ||
By the end of phase 4 we hope to have some useful test constructs that will allow us to perform motility assays and examine the biomaterial production efficiency. If we have time (and it is feasible) an extra 4 cloning steps will allow us to produce a complete construct with all our devices placed together; if we can integrate this successfully, we'll be able to perform system-level testing and maybe even produce some patterns on plates! | By the end of phase 4 we hope to have some useful test constructs that will allow us to perform motility assays and examine the biomaterial production efficiency. If we have time (and it is feasible) an extra 4 cloning steps will allow us to produce a complete construct with all our devices placed together; if we can integrate this successfully, we'll be able to perform system-level testing and maybe even produce some patterns on plates! | ||
All the constructs have been asigned codes and the schematic for production (assuming all goes well) has been finalised and can be accessed [[www.openwetware.org/wiki/Media:Strategy.doc | here]] | |||
= Protocols = | = Protocols = | ||
Revision as of 02:34, 18 August 2008
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Team Strategy
We have decided to split the protocols work into 5 sections
- Cloning Strategy - Tom & Chris
- PCR - (Tom & Chris)
- B.subtilis - James
- Calibration curves
- Testing
Mircoscope work - Krupa
1, 2 and 3 should be completed by monday morning so as to start some lab work. Microscope information requested to be sent to Martin by Friday.
Testing Constructs
Click here to see the test constructs
Critical Pathway
This diagram shows our critical pathway; the cloning steps we will undertake to produce our final constructs (and on the way, produce some valuable test constructs). We aim to re-use as many parts as possible to reduce the number of steps, but because of planned redundancy we often have multiple variants for steps. So for each addition of a promoter, there are actually four cloning procedures; one for each combination of the two promoters and RBSs we have ordered.
In total, we believe there are 101 cloning steps.
Without the sequences we ordered from Geneart, we can make a start by using the parts acquired from the Registry. These experiments are thus termed phase 0. Phase 1 requires some of our Geneart sequences - most notably the promoters/RBSs - and is focused on characterisation of the constitutive promoters. Phases 2 and 3 involve construction of test constructs to characterise our inducible promoters (which will lead our epsE and biomaterial genes) and phase 4 looks at light-inducible promoters.
By the end of phase 4 we hope to have some useful test constructs that will allow us to perform motility assays and examine the biomaterial production efficiency. If we have time (and it is feasible) an extra 4 cloning steps will allow us to produce a complete construct with all our devices placed together; if we can integrate this successfully, we'll be able to perform system-level testing and maybe even produce some patterns on plates!
All the constructs have been asigned codes and the schematic for production (assuming all goes well) has been finalised and can be accessed here
Protocols
Protocol Sandbox
General Format
Link to the protocols we need!
1.Transformation Protocols for integration vectors in Bacillus subtilis
2. Transformation Protocol for linear DNA in Bacillus subtilis
3.Transformation Protocol E.coli
5. PCR
6.Calibration Curves
- O.D.600 vs cell number
- Fluorescence vs GFPmut3b
7. Promoter-RBS Testing Protocols
- Constitutive Promoters and RBS
- pHyperspank Promoter and RBS
- Light Inducible Promoters and RBS
These will collapse into the names on the official wiki...
Protocol 1
The actual protocol
Protocol 2
Second protocol
Shopping List
Reagents and Materials
Link to the Shopping list for Reagents and Materials.
Geneart
Click here to see the Construct ordered from Geneart
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