NCBS Bangalore 2006
From 2006.igem.org
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What should we work on? | What should we work on? | ||
| - | *Group I: | + | *'''Group I''': |
**Develop a system by which Ecoli can sense two different colours. Red detection already exists, how can we pick up green independently? Can the two sensors be used to drive two different outputs? Try to couple these to X-Y chemotaxis. Problems: where can we quickly find a green sensor? | **Develop a system by which Ecoli can sense two different colours. Red detection already exists, how can we pick up green independently? Can the two sensors be used to drive two different outputs? Try to couple these to X-Y chemotaxis. Problems: where can we quickly find a green sensor? | ||
**Bacterial Fourier transform. Deliver some stimulus, e.g. light, in square pulses of a certain frequency. The output is the level of a reporter which can be correlated to frequency. Problems: how to make the amplitute of the output independent of the amplitute of the input signal, dependent only on the frequency? | **Bacterial Fourier transform. Deliver some stimulus, e.g. light, in square pulses of a certain frequency. The output is the level of a reporter which can be correlated to frequency. Problems: how to make the amplitute of the output independent of the amplitute of the input signal, dependent only on the frequency? | ||
**Bacterial signaling on wires and other patterns. Use a lawn of bacteria and some kind of template (e.g. a pattern of light) to activate certain cells. These cells can then talk to each other, transmitting a signal in defined patterns. Problems: for signalling around a ring, how to establish the initial asymmetry that makes transmission uni-directional? | **Bacterial signaling on wires and other patterns. Use a lawn of bacteria and some kind of template (e.g. a pattern of light) to activate certain cells. These cells can then talk to each other, transmitting a signal in defined patterns. Problems: for signalling around a ring, how to establish the initial asymmetry that makes transmission uni-directional? | ||
| - | *Group II: | + | *'''Group II''': |
**Using localized plasmids to detect the activity of a DNA-binding protein. (This is a research project in MT’s lab.) Introduce binding sites for CRP onto pole-localized plasmids; make a CRP-CFP fusion. When CRP is active, it will be pole localized; when it is inactive, it will be cytoplasmic. Problems: which pole-localized plasmid? How to ensure that additional binding sites and fusion proteins don’t perturb cell function? | **Using localized plasmids to detect the activity of a DNA-binding protein. (This is a research project in MT’s lab.) Introduce binding sites for CRP onto pole-localized plasmids; make a CRP-CFP fusion. When CRP is active, it will be pole localized; when it is inactive, it will be cytoplasmic. Problems: which pole-localized plasmid? How to ensure that additional binding sites and fusion proteins don’t perturb cell function? | ||
**X-Y chemotaxis. We would like +/- control on two independent axes. This would require 4 different chemical gradients, two in each orthogonal direction. Suggestion: we could have default taxis in the ‘-' orientation, and active taxis in the ‘+’ orientation. Problems: how to make the cell ‘stand still’ in this configuration? Active feedback? | **X-Y chemotaxis. We would like +/- control on two independent axes. This would require 4 different chemical gradients, two in each orthogonal direction. Suggestion: we could have default taxis in the ‘-' orientation, and active taxis in the ‘+’ orientation. Problems: how to make the cell ‘stand still’ in this configuration? Active feedback? | ||
| Line 50: | Line 50: | ||
**Synchronizing cell cycles. Use a cell cycle-dependent promoter (e.g. cya in Ecoli) to drive a cell-cell signal. Use the signal to modulate cell cycle in the receiver cell. This should allow synchronization of the population. Problems: How to modulate the cell cycle? | **Synchronizing cell cycles. Use a cell cycle-dependent promoter (e.g. cya in Ecoli) to drive a cell-cell signal. Use the signal to modulate cell cycle in the receiver cell. This should allow synchronization of the population. Problems: How to modulate the cell cycle? | ||
| - | *Group III: | + | *'''Group III''': |
**Magnetic paintbox. Combine magnetotaxis with 3 colour detection to make a colour pattern generator. Problems: cannot reconstitute magnetotaxis in Ecoli. | **Magnetic paintbox. Combine magnetotaxis with 3 colour detection to make a colour pattern generator. Problems: cannot reconstitute magnetotaxis in Ecoli. | ||
**Synchronizing repressilators. This is based on a suggestion by Strogatz and Elowitz. Problems: the repressilator is an unreliable oscillator. | **Synchronizing repressilators. This is based on a suggestion by Strogatz and Elowitz. Problems: the repressilator is an unreliable oscillator. | ||
**An early warning chemotaxis system for UV damage. (This was suggested by Mrinalini Puranik.) A beam of UV light slowly moves over a bacterial lawn. Cells under UV suffer DNA damage and upregulate their repair mechanisms. Some of these cells actively move away from the light toward other parts of the plate, and trigger the repair response in neighboring cells. Problems: can we get unidirectional motion? How to sense UV damage? What pathways should we upregulate in receiver cells? | **An early warning chemotaxis system for UV damage. (This was suggested by Mrinalini Puranik.) A beam of UV light slowly moves over a bacterial lawn. Cells under UV suffer DNA damage and upregulate their repair mechanisms. Some of these cells actively move away from the light toward other parts of the plate, and trigger the repair response in neighboring cells. Problems: can we get unidirectional motion? How to sense UV damage? What pathways should we upregulate in receiver cells? | ||
**Bacterial segregation by attractant/repellent strategy. Two populations of cells will be mixed. One synthesizes an attractant for its own kind; another synthesizes a repellent for the first kind. Together, this would cause cells to segregate, leading to Turing-like patterns. Suggestion: make use of molecules which can behave both as attractants and repellents depending on concentration (e.g. L-Lysine). Problems: we can make sure that different populations have distinct attractants, but we do not have control over repellents. Is a universal repellent sufficient to achieve the desired effect? | **Bacterial segregation by attractant/repellent strategy. Two populations of cells will be mixed. One synthesizes an attractant for its own kind; another synthesizes a repellent for the first kind. Together, this would cause cells to segregate, leading to Turing-like patterns. Suggestion: make use of molecules which can behave both as attractants and repellents depending on concentration (e.g. L-Lysine). Problems: we can make sure that different populations have distinct attractants, but we do not have control over repellents. Is a universal repellent sufficient to achieve the desired effect? | ||
| + | |||
| + | [[Feb 11]], [[2006]] | ||
| + | |||
| + | Each group has picked a single project upon which to focus. The task now is to think about intermediate goals, and chart out a research schedule for the coming month. The important aspect of each project is not the end result, but the design principles that will ensure a successful outcome. The question is: how to do control and validation experiments, and how to design intermediate constructs, that will maximize the chances of success. What we are developing here is not a product, but a process. The three projects span the range of construct complexity: one group requies a single regulated gene, while another is assembling a network of several genes and promoters. The simplest construct is most likely to do what we intend it to do; the more complex ones will require some coaxing. The projects also engage traditional biological questions to different extents: some aspects are interesting from a pure science perspective, while others are useful to engineers. | ||
| + | |||
| + | *'''Group I''': | ||
| + | Project: Regulation of X-Y chemotaxis. Use two attractants whose receptors are inducible (serine with Tar, aspartate with Tsr). These define two orthogonal axes. Use a default attractant along a diagonal to establish '-' movement. Immediate goals: To establish substrate geometries upon which multiple reproducible, stable, quantitative chemical gradients can be established; to validate default chemotaxis in the absence of MCP receptors. | ||
| + | |||
| + | *'''Group II''': | ||
| + | Project: Synchronization of bacterial cell cycles. Use a cell cycle-dependent promoter to drive a LuxI-LuxR based cell-cell signal. Use regulation of replication initiator DnaA to modulate cell cycle in receiver cells. Immediate goals: To determine if candidate promoters oscillate; to regulate DnaA levels. | ||
| + | |||
| + | *'''Group III''': | ||
| + | Project: Analysis of stress responses in biofilms. Connect stress response system to a lac based bistable GFP reporter. This allows transient expression at a stress-response promoter to trigger persistent expression of GFP. Immediate goals: To standardize biofilm assay; to design other experiments in which such a construct would be useful, especially using a cell-sorter. | ||
| + | |||
| + | [[Feb 18]], [[2006]] | ||
| + | |||
| + | Each group has submitted a DNA-level design for their construct. While these constructs are being synthesized, the groups should now work on measurement assays and apparatus. | ||
| + | |||
| + | *'''Group I''': | ||
| + | Project: Regulation of X-Y chemotaxis. Ecoli strain UU1250 has 5 MCP-type chemotaxis receptors knocked out. The Tar and Tsr receptors (taxis towards Aspartate and Serine) will be put back on inducible promoters, Plac and Ptet. Chromosomal copies of lacI and tetR will be moved into strain UU1250 from strain Z1 by P1 transduction. Immediate goals: To test gradients in solid agar substrate; use various geometries and dyes to monitor time-dependence of gradients. Reservoirs and sinks seem a promising strategy. | ||
| + | |||
| + | *'''Group II''': | ||
| + | Project: Synchronization of bacterial cell cycles. Complex construct is broken into simpler pieces to test the following aspects: (1) Does Pcya oscillate? Monitor LuxI-CFP fusion. (2) How much does oscillation attenuate: Use Plux and YFP. (3) How does DnaA sequestration affect replication? Use Plac to desequester DnaA. (4) How does DnaA affect Pcya? Use combination of constructs to test this. And so on. Immediate goals: To build a chemostat in which cell densities can be regulated, either by manual feedback or by limiting nutrients. | ||
| + | |||
| + | *'''Group III''': | ||
| + | Project: Analysis of stress responses in biofilms. Stress-response monitor has been designed. Use RFP to monitor immediate SOS promoter activation, and GFP to monitor history of activation. | ||
| + | Immediate goals: To standardize biofilm assay; to test TMG thresholds in solid medium. | ||
Revision as of 21:42, 6 July 2006
Contents |
The Living Networks Wiki
May 11, 2024
The NCBS iGEM team grew out of an interdisciplinary workshop which ran through the spring of 2006. Participants included students, faculty, and industry researchers; physicists, biologists, computer scientists, and engineers; lawyers, educators, and entrepreneurs. Our discussions on genetic networks, synthetic biology, open-source ideas, and industrial applications, are documented on the workshop [http://www.ncbs.res.in/~faculty/ssb website].
The team
Please feel free to change your titles. We'll order new business cards every week.
- Mukund: Chief Mentor and Brainstormer (thattai@ncbs.res.in)
- Adil: Chief Critic and Voice of Reason (adil@ncbs.res.in)
- Aparna: Chief Mind-Mapping Officer (aparna@ncbs.res.in)
- Ashesh: Head of Getting-Things-To-Work Department (ashesh@ncbs.res.in)
- Dhanya: Wild Ideas Consultant (dhanya@ncbs.res.in)
- Krithiga: Team Whip and Strategist (krithiga@ncbs.res.in)
- Ruchi: Head of Enthusiasm Department (mallik@ncbs.res.in)
- Sugat: Chief Technical Officer and Head of Big-Picture Department (sugat@ncbs.res.in)
The projects
- X-Y regulation of bacterial chemotaxis
- Synchronization of bacterial cell cycle
- Monitoring long-term effects of UV exposure
Lab notes
What should we work on?
- Group I:
- Develop a system by which Ecoli can sense two different colours. Red detection already exists, how can we pick up green independently? Can the two sensors be used to drive two different outputs? Try to couple these to X-Y chemotaxis. Problems: where can we quickly find a green sensor?
- Bacterial Fourier transform. Deliver some stimulus, e.g. light, in square pulses of a certain frequency. The output is the level of a reporter which can be correlated to frequency. Problems: how to make the amplitute of the output independent of the amplitute of the input signal, dependent only on the frequency?
- Bacterial signaling on wires and other patterns. Use a lawn of bacteria and some kind of template (e.g. a pattern of light) to activate certain cells. These cells can then talk to each other, transmitting a signal in defined patterns. Problems: for signalling around a ring, how to establish the initial asymmetry that makes transmission uni-directional?
- Group II:
- Using localized plasmids to detect the activity of a DNA-binding protein. (This is a research project in MT’s lab.) Introduce binding sites for CRP onto pole-localized plasmids; make a CRP-CFP fusion. When CRP is active, it will be pole localized; when it is inactive, it will be cytoplasmic. Problems: which pole-localized plasmid? How to ensure that additional binding sites and fusion proteins don’t perturb cell function?
- X-Y chemotaxis. We would like +/- control on two independent axes. This would require 4 different chemical gradients, two in each orthogonal direction. Suggestion: we could have default taxis in the ‘-' orientation, and active taxis in the ‘+’ orientation. Problems: how to make the cell ‘stand still’ in this configuration? Active feedback?
- Bacterial log book. If we have a single cell moving around a plate, can we reproduce its path? We would have to use DNA sequence rather than proteins, because the latter limits us to a few points. Use lambda insertion or transposon insertion; kill cells that return to the same point twice. Problems: The insertion mechanism is completely undefined.
- Synchronizing cell cycles. Use a cell cycle-dependent promoter (e.g. cya in Ecoli) to drive a cell-cell signal. Use the signal to modulate cell cycle in the receiver cell. This should allow synchronization of the population. Problems: How to modulate the cell cycle?
- Group III:
- Magnetic paintbox. Combine magnetotaxis with 3 colour detection to make a colour pattern generator. Problems: cannot reconstitute magnetotaxis in Ecoli.
- Synchronizing repressilators. This is based on a suggestion by Strogatz and Elowitz. Problems: the repressilator is an unreliable oscillator.
- An early warning chemotaxis system for UV damage. (This was suggested by Mrinalini Puranik.) A beam of UV light slowly moves over a bacterial lawn. Cells under UV suffer DNA damage and upregulate their repair mechanisms. Some of these cells actively move away from the light toward other parts of the plate, and trigger the repair response in neighboring cells. Problems: can we get unidirectional motion? How to sense UV damage? What pathways should we upregulate in receiver cells?
- Bacterial segregation by attractant/repellent strategy. Two populations of cells will be mixed. One synthesizes an attractant for its own kind; another synthesizes a repellent for the first kind. Together, this would cause cells to segregate, leading to Turing-like patterns. Suggestion: make use of molecules which can behave both as attractants and repellents depending on concentration (e.g. L-Lysine). Problems: we can make sure that different populations have distinct attractants, but we do not have control over repellents. Is a universal repellent sufficient to achieve the desired effect?
Each group has picked a single project upon which to focus. The task now is to think about intermediate goals, and chart out a research schedule for the coming month. The important aspect of each project is not the end result, but the design principles that will ensure a successful outcome. The question is: how to do control and validation experiments, and how to design intermediate constructs, that will maximize the chances of success. What we are developing here is not a product, but a process. The three projects span the range of construct complexity: one group requies a single regulated gene, while another is assembling a network of several genes and promoters. The simplest construct is most likely to do what we intend it to do; the more complex ones will require some coaxing. The projects also engage traditional biological questions to different extents: some aspects are interesting from a pure science perspective, while others are useful to engineers.
- Group I:
Project: Regulation of X-Y chemotaxis. Use two attractants whose receptors are inducible (serine with Tar, aspartate with Tsr). These define two orthogonal axes. Use a default attractant along a diagonal to establish '-' movement. Immediate goals: To establish substrate geometries upon which multiple reproducible, stable, quantitative chemical gradients can be established; to validate default chemotaxis in the absence of MCP receptors.
- Group II:
Project: Synchronization of bacterial cell cycles. Use a cell cycle-dependent promoter to drive a LuxI-LuxR based cell-cell signal. Use regulation of replication initiator DnaA to modulate cell cycle in receiver cells. Immediate goals: To determine if candidate promoters oscillate; to regulate DnaA levels.
- Group III:
Project: Analysis of stress responses in biofilms. Connect stress response system to a lac based bistable GFP reporter. This allows transient expression at a stress-response promoter to trigger persistent expression of GFP. Immediate goals: To standardize biofilm assay; to design other experiments in which such a construct would be useful, especially using a cell-sorter.
Each group has submitted a DNA-level design for their construct. While these constructs are being synthesized, the groups should now work on measurement assays and apparatus.
- Group I:
Project: Regulation of X-Y chemotaxis. Ecoli strain UU1250 has 5 MCP-type chemotaxis receptors knocked out. The Tar and Tsr receptors (taxis towards Aspartate and Serine) will be put back on inducible promoters, Plac and Ptet. Chromosomal copies of lacI and tetR will be moved into strain UU1250 from strain Z1 by P1 transduction. Immediate goals: To test gradients in solid agar substrate; use various geometries and dyes to monitor time-dependence of gradients. Reservoirs and sinks seem a promising strategy.
- Group II:
Project: Synchronization of bacterial cell cycles. Complex construct is broken into simpler pieces to test the following aspects: (1) Does Pcya oscillate? Monitor LuxI-CFP fusion. (2) How much does oscillation attenuate: Use Plux and YFP. (3) How does DnaA sequestration affect replication? Use Plac to desequester DnaA. (4) How does DnaA affect Pcya? Use combination of constructs to test this. And so on. Immediate goals: To build a chemostat in which cell densities can be regulated, either by manual feedback or by limiting nutrients.
- Group III:
Project: Analysis of stress responses in biofilms. Stress-response monitor has been designed. Use RFP to monitor immediate SOS promoter activation, and GFP to monitor history of activation. Immediate goals: To standardize biofilm assay; to test TMG thresholds in solid medium.
