CHE.496/2009/Responses/a8

Biological Machines

• Discussion leader: Rohini

Rohini's Response

Designing Biological Systems

• Main Point- review progress made in the field of synthetic biology
• Synthetic biology- seeks to modify or mimic biological systems by using natural cellular components as well as generate unnatural chemical systems
• Overall, the construction of systems is the product of iterative cycles of computer modeling, biological assembly and testing

The four developments that synthetic biologists have to wait for before engineering whole organisms are:

• inexpensive DNA synthesis, rapid and inexpensive DNA sequencing, collection of components that are well-characterized, adoption of engineering approaches

• Role mathematical modeling plays in designing gene circuits:

Ex.- repressilator, use of a quantitative model of the system was important in determining the parameters in which the system is oscillatory

• Two ways of achieving bistability in a network: creating a positive feedback loop and creating mutual repression

• Synthetic switches in both pro/eukaryotes can be mediated via RNA devices (engineered riboregulators)
• By introducing specific mutations in portions of the RNA sequences, the switching behavior of the system can be altered
• Certain evolutionary precursors gave rise to diverse types of organisms because they had design features that were easier for evolution to work

• Challenge in engineering bacteria- the cell wall makes it impossible for cells to communicate by cell contact

• Example of a synthetic circuit that has implemented artificial cell-cell interaction:

Pulse generator- the receiver cell responds to the signal sent by the sender cell with a pulse of GFP. The system demonstrated that receiver cells respond to a varying response to sender cells based on the rate of increase of inducer concentration and their distance from the sender cell

• Example of engineered signaling pathway in a system:

-Reprogramming of an allosteric protein signaling switch in yeast

• Metabolic engineering-integrating new pathway into a cell to synthesize a key metabolite.
• Example: synthesis of artemisinin, component in antimalarial drug

• Two strategies for studying the simplest organism:

1) Top-down approach: organisms are simplified further through the removal of nonessential genetic elements 2) Bottom-up approach: synthesis of artificial cells is attempted, component by component

• Problems with using PCR to synthesize DNA:

-too laborious, high rate of errors and repair is done by site-directed mutagenesis -“copy, cut and paste” manner using polymerases, restriction endonucleases and ligases

• Currently used technique for synthesizing DNA:

-coupling of large scale oligo synthesis on microfluidic microarrays with mismatch error correction and a single step polymerase assembly to the build the gene

• Proposed engineering approaches to make work within the field of synthetic biology easier:

-use of well-characterized parts, standardization, abstraction

Biology by design: reduction and synthesis of cellular components and behavior

• The article discusses two types of work that goes on within the field of synthetic biology. It mentions that the field encompasses the design and construction of new biological parts, devices and systems. But, it also discusses the re-design of existing, natural biological systems for useful purposes such as the development of therapeutics, renewable energies.

• The biggest challenge that scientists face in creating simple biological systems is predicting the system behavior

• Steps for engineering gene circuits:

-Determine the design goal, pick suitable host organisms, identify necessary parts, model, explore circuit dynamics, implement/test/debug

• Florescent protein helps in monitoring synthetic circuits by monitoring in vivo protein levels

• Microfluidics-manipulating small amounts of fluids using microsized channels help in the integration of complex chemical or biological procedures into a single process that is faster

• Reverse sorting:cells scanned at a high flow rate until a fluorescent cell is detected and then flow is stopped and reversed allowing the cell to be measured and diverted into a collection tube. The purpose is to isolate rare cells or to make multiple measurements of the cell.

Rohini Manaktala 16:30, 11 March 2009 (EST)

Thaddeus's Response

Designing Biological Systems

• Purpose to summarize the work done in synthetic biology.
• Early circuits were inspired by naturally existing biological or nonbiological human designed ciruits
• Circuits created include repressilator, toggle switch, feedback loop
• natural systems often contain many more elements than synthetic ones
• Toggle switches can give memory to a system
• Switches can be in RNA form
• Properties of cell can affect how strongly evolution can act on organism
• Multicellular systems have been created using small molecule signals
• Quorum sensing pulse generator was created in yeast.
• Signal cascades would allow from more complex switches.
• Metabolic engineering must account fo the cells resource drain.
• Novel biosensors have been engineered.
• Synthetic genome may allow for use of unnatural amino acids.

Biology by design: Reduction and synthesis of cellular components and behavior link

• Ability to make a gene relies on ability to fabricate and knowledge of what to fabricate.
• Synthetic biology has been made possible by
  • Improved DNA synthesis.
  • ability to design parts/computer modelling
  • Circuit engineering based on modelling reduces trial and error in design.
  • Synthesis of more reporter genes has allowed better characterization of circuits
• Techniques have been developed to follow single molecules in vivo.
• Microfluidics will decrease debugging time.

Synthetic biology will be useful for many applications

• Gentler chemical synthesis
• Drug production
• Research in life sciences
• Living drugs
• Energy production, converting cellulose to ethanol

Thaddeus Webb 01:52, 12 March 2009 (EDT)

Joe Bozzay's Response

Designing biological systems

• Article gives a summary of the advances in synthetic biology
• Some synthetic circuits are analogous to previously existing circuits, but many have the goal of being kept simple.
• Synthetic oscillators and switches: demonstrate importance of mathematical modeling and important to controlling cyclic events in the cell
• Can use feedback, repression, and RNA mutations to control the stability and switching of the system.
• Artificial cell-cell interaction systems: pulse generators and band detectors and experiments reveal the role of diffusion gradients in cell-cell signaling.
• Engineering signal transduction: signal transduction cascades that regulate pathways through modifications or ligand binding. Further study will allow the development of more complicated network designs.
• The key to understanding fast responding genetic circuits lies in understanding protein manipulation and enzyme cascades
• Biosensors: using computer aid to design new proteins to accomplish a specific purpose in the pathway.
• Advances: DNA synthesis is cheaper and faster. The registry of biological parts has provided characterization. Synthetic genomes could allow the integration of unique amino acids.

Biology by design: reduction and synthesis of cellular components and behavior

• Due to the increased understanding of the cellular systems and the advances in technology, the focus of synthetic biology is to apply the knowledge to accomplish a purpose.
• The design and production of biological parts is a necessity.
• Improved DNA synthesis has improved the field and advanced past the “cut and paste” DNA manipulation methods previously used.
• Abstraction hierarchy: DNA->parts->circuits->cells->populations
• Mathematical formulations and models have advanced and optimized experimentation methods and the design of biological parts.
• The ability to “debug” through reporter genes.
• Still difficult to predict system behavior.
• List for engineering genetic circuits: pick a design goal, pick host, identify parts, model, test, and debug. It is rare the circuit will work the first time, so be prepared to loop through the last few procedures.
• Applications: synthesis of needed chemicals in high yields, like artemisinic acid (drug), insulin, and antibodies. Also will be useful for energy production.

Joe Bozzay 15:24, 12 March 2009 (EDT)

Maria

Designing Biological systems

• The article opens by discussing modeling- will we be doing this in the summer?
• Repressilator:
  • 3 repressors in a loop
• noise can be useful (?)
• Rhythms: can simplify and study systems
• bistability: genetic toggle switch w/ memory
  • positive feedback or mutual repression
  • non-linear system
  • 2 genes express each others’ repressor
  • system will stay at one stable pt until stimulated to go to next
• DNA damage:
  • repressor was cleaved with DNA damage and so circuit could be made to produce output (biofilm) in response to DNA damage.
• positive feedback:
  • tetracycline switched cells to “on”
  • bi modal: “on” v. “off”
• noise-based switch
• regulation with antisense
  • regulates RNA
  • no byproducts
  • fast
• cell-cell
  • pulse generator: gfp is output
  • responds to specific concentrations ( can work spatial-temporally)

Biology by design

• algoritms and more powerful processors improve computational design
• design parts invivo can be manipulated
• can identify and manipulate molecules
• secondary structure prediction of RNA- could help improve control switches
• gene expression models are often simplified
• use of genetic manipulation can avoid toxic by-product production from manufacture
  • production of antimilaria drugs from yeast
• metabolic engineering: production of non-protein product (ie artemisinic acid)

• Maria Fini 18:23, 12 March 2009 (EDT):