20.109(S09)/PinkWF: Difference between revisions
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* Common intracellular signal carrier for transcription-based devices. BBa_F2620 output is PoPs, so we'd have to have a trigger with PoPs input. | * Common intracellular signal carrier for transcription-based devices. BBa_F2620 output is PoPs, so we'd have to have a trigger with PoPs input. | ||
* No in vivo technique for measuring PoPs directly, but can do it indirectly (look at endy paper above) | * No in vivo technique for measuring PoPs directly, but can do it indirectly (look at endy paper above) | ||
*Abstraction hierarchy: http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi | |||
Gene Gateway (need to figure this out) | Gene Gateway (need to figure this out) |
Revision as of 01:08, 12 May 2009
20.109 Research Proposal for Derek Ju and Alvin Chen
Project Overview
Our goal is to rewire a bacterium to "miniprep" itself, when given some sort of an external stimuli (such as sensing of dense cell growth). This project will serve the following purposes:
- Make the research process easier, cheaper, and more efficient for scientists working with bacteria.
- Most laboratory strains have been genetically modified for scientific use; however our bacteria would be the first to be modified to do an experiment on itself.
- Self-lysing of cells when dense cell growth is encountered - less chance for environmental contamination if genetically engineered bacteria escape from the lab
- Serve as an example of a way synthetic biology can be used for cellular control
- Be an application of using irreversible switches within genetic circuits
- Be an example of sensors being built into bacteria
Background
Cell-cell signaling:
- Need two devices: Sender device and Reciever device. Current devices are based on Lux system of V. Fischeri or analogs. http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=Signalling
- Sender: BBa_F1610
- Reciever: BBa_F2620 http://www.nature.com/nbt/journal/v26/n7/full/nbt1413.html. Characterization of this part by Drew Endy
Polymerases Per Second (PoPs)
- Standard unit for signal via which information is transmitted in transcription-based logic devices. (http://openwetware.org/wiki/PoPS.
- Common intracellular signal carrier for transcription-based devices. BBa_F2620 output is PoPs, so we'd have to have a trigger with PoPs input.
- No in vivo technique for measuring PoPs directly, but can do it indirectly (look at endy paper above)
- Abstraction hierarchy: http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi
Gene Gateway (need to figure this out)
- Input would be an amount of PoPs generated from the cell quorum sensor
- How do we set a threshold for PoPs toleration amount?
- maybe simple constitutive inhibitor production until it's overwhelmed
- Need to measure inhibitor production and how to calibrate this
- maybe simple constitutive inhibitor production until it's overwhelmed
- Output leads to generation of lysozyme AND RNase (anything else)
- IGEM 2004 polkadorks (http://parts.mit.edu/wiki/index.php/IAP2004:Polkadorks)
Biobricks
- A lot of parts are available for us to use already
- May need to add new parts (hopefully)
- Need to characterize these parts
- Not good for fusion proteins (do we need any?) but can use modified Silver standard for this
Leucine Zipper
- Clump cells together (pros/cons to this?)
Reporters
- attaching GFP, RFP etc to gene circuits would allow for quick, easy determination of whether it's working
- Good for debugging
- Should only be for experimental design, final product should not have this
Lysozyme Gene (which?)
RNase A Gene (which?)
How to isolate only DNA?
- Can we easily isolate nucleotides since RNA isn't present anymore?
- Some kind of universal proteolysis? How to get rid of residues?
- How to isolate plasmid DNA vs genomic DNA? Can we find a way to do both?
- Maybe if you get rid of enough proteins/RNA then u can just use the mixture to do the next reaction (transformation, digestion) with some type of efficiency, but this would have to be studied
Characterization of parts
- Efficiency of expression of lysosome and RNase
- Efficiency of gateway
- Efficacy of RNA degradation
- Efficacy of transformations with this mixture OR purity of plasmid if we decide to purify
How does miniprep work?
- What does each buffer, spin column, etc do?
- How much does it cost?
How does genomic DNA isolation work??
Research Problems and Goals
Competing Technologies: Qiagen miniprep, Berkeley clonebots Safety: Don't know toxic effects of inserting these genes, but it should make it safer by having a self-aptosis mechanism
Project Details and Methods
In order to complete our project, we will attempt to incorporate several new genes into E. coli, which will encode for various proteins.
- We will insert a gene encoding for a binding protein that will be attached to the membrane of the bacteria. This protein will most likey contain leucine zippers and as a result the bacteria will lump together in culture.
- The attachment of the binding proteins to eachother will indicate clumping of cells, which will release a signal to toggle a "genetic switch". This switch will be a collection of genes, which when combined creatively, can have an input and output.
- The activation of the genetic switch will lead to expression of a gene encoding for lysozyme, which will lyse the cell.
- The activation of the genetic switch will lead to expression of a gene encoding for RNAse A, which will degrade the RNA in the cell.
Project Overview (abstract)
- Cells secrete chemical --> cells detect high levels of extracellular chemical --> Activate gateway --> produce Lysozome & RNase
Gene Pathway (abstract)
- Quorum signal sender device ---3OC6HSL---> Quorum signal reciever ---PoPs---> Genetic Gateway ---ON--->Lysozome & RNase production
Predicted Outcomes
Don't know:
- secretion efficiency of lysosome needed to lyse cell
- same for RNase to degrade RNA
- How to calibrate the threshold for PoPs to activate gateway (usually we wait for cell growth to reach a certain density until we miniprep it)
- How do the modifications affect the cell
- How to isolate DNA, and further how to isolate genomic from plasmid (make sure this is not harder than using a miniprep kit)
Resources Needed
- Appropriate DNA sequences for genes, signal sequences, etc
- Standard plasmid for transformation into E. coli, such as pSK1A2
- Some of the DNA for the genetic switches can be retrieved from the Biobricks foundation
References
- http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html
- Proteins bind to promoter regulatory sites to repress or activate expression. This results in two stable states: proteins (repressor) repress their own repressor, so they will remain stably expressed. But, switching is controllable switching: dominant repressor (state) can be de-activated with addition of inducer (IPTG, thermal shock, etc). This allows expression of the other repressor, and enables controllable state change of the system
- http://www.ncbi.nlm.nih.gov/pubmed/3289117?dopt=Abstract
- Leucine zippers are a common structures that have key functions within binding proteins.