CHE.496/2009/Responses/a6

Practical applications

• Discussion leader: Brandon

Thaddeus's Response

Advances in synthetic biology: on the path from prototypes to applications

• This article observes the capabilities and potentials of genetic circuits
• focus of synthetic biology started on transcriptional cascades
• Longer cascades are more sensitive and have a narrower switching region between on and off
• Feed forward mechanisms cause the steady state output to remain a function of the input.
• Feedback systems can be used to create bistable toggle switches
• Combination of above components have yet to reach potential but will be limited by cellular mechanisms.
• Cell-cell communication can coordinate multicellular behaviors.
• Multicellular systems can be given spatial components.
• Models can be used to consider kinetics and network connectivity.
• Directed evolution is a tool to optimize less understood systems through random mutagenesis.
• Useful because outlines functional components of genetic circuits.

Molecular switches for cellular sensors

• Describes RNA based switching mechanisms
• Nucleic acids have catalytic capabilities.
• Nucleic acids can have regulatory functions.
• Nucleic acids can act as sensors.
• Combining above functions allows creation of RNA switches which detect molecules and change translational outputs of cell.
• Used to detect theophylline and give a measurable response.
• Demonstrated specificity and controllable variable sensitivity.
• Showed ability to combine multiple RNA sensors without cross interaction.
• RNA switches could be useful for us as a simpler way to make novel detectors.

Thaddeus Webb 02:36, 26 February 2009 (EST)

Rohini's Response

Molecular Switches for Cellular Sensors

• Synthetic biology- design cellular systems programmed to complete specific tasks
• Metabolic engineering- reprograms a cell to produce a valuable compound, i.e. pharmaceutical
• Interesting fact: (S)-reticuline, an intermediate metabolite in opium poppy is used to make anticancer and antimalarial drugs. It is very difficult to interrupt its metabolic pathway because different intermediates accumulate and the interference could actually inhibit enzymatic steps earlier in the metabolic pathway.
• Author’s goal: To control the production process in a microorganism using genetic-engineering tools and artificially constructed pathways.
• Application: Reprogram a diseased or problematic cell through “intelligent molecular therapeutics.” This can be achieved by designing molecules that could potentially identify the cell that they are in and perform an action based on that identification. (Ex. Biosensors)

• Nucleic acids perform various functions:
• 1) Exhibit catalytic activity- perform cleavage/ligation reactions
• 2) Act as regulatory elements- “trans-acting RNA” molecules which do not code for any protein regulate protein production; “cis-acting RNA” molecules interact with other biomolecules to regulate the rates of transcription, decay and translation.
• 3) Function as sensors- detect and identify other molecules. [RNA or DNA molecule folds back onto itself to create a binding site for a protein molecule, i.e. caffeine, drugs, lipids in cell membranes].
• -Advantage: can produce molecular sensors outside of cells (in vitro)
• -Process: Random pool of nucleic acids --> transform into corresponding RNAs --> incubate with the molecule the sensor is going to recognize--> extract RNAs that bind to the target molecule --> repeat steps again [End product- very selective, high-affinity group of aptamers which are nucleic acid structures that bind to the target]
• -Example of a sensor, when a green fluorescent protein is excited by a laser the cell emits a green light

• Put anti-switches in cells to see if the biosensors will function. (Ex. Aptamer that recognizes theophylline, had no switch effect with caffeine thereby proving sensor domains differentiate between very similar molecules).

• Future- design a chip-based diagnostic device that could detect the presence of proteins, sugars and other small molecules in a blood, urine or saliva sample.

I really enjoyed reading this article. I thought the experiment performed by the Caltech group was quite interesting. Maybe for our VGEM project, we could create a biosensor for a specific ligand that can be used in a diagnostic test.

Advances in synthetic biology: on the path from prototypes to applications

• Synthetic biology- engineer genetic elements to create more complex functions
• Genes are arranged in series and each gene product regulates the expression of one target
• Evolution has optimized the regulatory interactions between network elements in cascades

Challenge- altering the kinetics of individual elements so that they function correctly within the context of the new network

• Feed-forward motifs- have a master regulatory gene that regulates target genes through multiple non-circulatory pathways. (steady-state output remains a f(x) of the input)
• Regulatory feedback- has more refined properties (steady-state output is not a f(x) of the input)
• Application of synthetic systems- pharmaceutical products

Challenge- its hard to develop a mathematical model representing the system’s behavior. However, running a sensitivity analysis would be a more accurate computational approach accounting for the different kinetics rates/trials.

Rohini Manaktala 15:12, 25 February 2009 (EST)

Patrick Gildea's Response

• Advances in synthetic biology: on the path from prototypes to applications
  • problems with uvaAnywhere
• Molecular Switches for Cellular Sensors
  • problems with uvaAnywhere vpn...
• Patrick Gildea 18:57, 25 February 2009 (EST):

Maria’s response=

• identify integral proteins that influence a behavior & interactions
• nucleic acids can be used to govern function (expression)
• functions:
  • catalytic activity: can cut other RNA’s, can ligate nucleic acids
  • regulatory function: “trans-acting RNA” aka antisense: binds to mRNA
    • some impede translation
    • some increase the decay rate of mRNA: flag for distruction
  • cis-acting:
    • part of mRNA
    • steam loop (not coded as protein): affects transcription/ translation/ decay of mRNA
  • sensors for small molecules (caffeine, lipids…)
    • simpler than protein-ligand model
• through trial and error, you can find RNA’s(aptamers) to bind to a specific ligand
• contain different domains: output domain controls the production of a protein.
• antiswitch is digital and threshold- dependent.

• and /or logic capabilities

Questions/comments for us:

• could we use RNA to catalyze reactions? (increase or decrease RNA being translated?)
• what about regulation of the destruction of RNA post- translation?
• examples?
• Maria Fini 18:21, 26 February 2009 (EST):

Joe’s response

Advances in synthetic biology: on the path from prototypes to applications

• Can create behaviors by integrating genetic elements into complex function circuits
• Cascades have a steady state output that is a correlating function of the input see the MAPK pathway
• Cascades also possess similar noise attenuation for high and low outputs, which shows a certain filtering capability
• Can make more sensitive circuits by lengthening cascades
• Utilize feedback to make switches
• Building new circuits is costly and time consuming.
• De novo synthesis and error correction techniques should speed the development
• Can use models to optimize network

Molecular switches for cellular sensors

• Nucleic acids (like found in RNA/DNA) possess many capabilities for molecular switches
• Nucleic acids can act as catalysts through cleaving and ligations
• Nucleic acids can also regulate reactions with the cis and trans RNA isomers
• Can also act as sensors through florescence
• Allows for specific binding and the ability to control sensitivity –> see theophylline experiment
• Ultimately these capabilities will be manifested in a nanochip (lab-in-a-chip)

Joe Bozzay 18:24, 26 February 2009 (EST)