Repressilator
Encyclopedia
The repressilator is a synthetic genetic regulatory network reported in a paper by Michael Elowitz
and Stanislas Leibler. This network was designed from scratch to exhibit a stable oscillation which is reported via the expression of green fluorescent protein
, and hence acts like an electrical oscillator system with fixed time periods. The network was implemented in Escherichia coli
using standard molecular biology methods and observations were performed that verify that the engineered colonies do indeed exhibit the desired oscillatory behavior.
, such that each gene represses the next gene in the loop, and is repressed by the previous gene. In addition, green fluorescent protein
is used as a reporter so that the behavior of the network can be observed using fluorescence microscopy.
The design of the repressilator was guided by two simple mathematical models, one continuous and deterministic and the other discrete and stochastic.
These models were analyzed to determine the values for the various rates which would yield a sustained oscillation. It was found that these oscillations were favoured by strong promoters coupled to efficient ribosome binding sites, tight transcriptional repression (low 'leakiness'), cooperative repression characteristics, and comparable protein and mRNA decay rates.
This analysis motivated two design features which were engineered into the genes:
First, to decrease leakiness the promoter regions were replaced with a tighter hybrid promoter which combined the λ PL promoter with LacL and TetR operator sequences.
Second, to reduce the disparity between the lifetimes of the repressor proteins and the mRNAs, a carboxy terminal tag based on the ssRA RNA sequence was added at the 3' end of each repressor gene. This tag is recognized by proteases which target the protein for degradation.
encoding the repressilator, and the higher copy reporter, which were used to transform a culture of Escherichia coli
.
which shows that genetic regulatory networks which perform a novel desired function can be designed and implemented. Further, this experiment gives new appreciation to the circadian clock found in many organisms, as they perform much more robustly than the repressilator.
Michael Elowitz
Michael B Elowitz is a biologist and professor of Biology, Bioengineering, and Applied Physics at the California Institute of Technology, and investigator at the Howard Hughes Medical Institute. In 2007 he was the recipient of the Genius grant, better known as the MacArthur Fellows Program for the...
and Stanislas Leibler. This network was designed from scratch to exhibit a stable oscillation which is reported via the expression of green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
, and hence acts like an electrical oscillator system with fixed time periods. The network was implemented in Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
using standard molecular biology methods and observations were performed that verify that the engineered colonies do indeed exhibit the desired oscillatory behavior.
Design
The repressilator consists of three genes connected in a feedback loopFeedback
Feedback describes the situation when output from an event or phenomenon in the past will influence an occurrence or occurrences of the same Feedback describes the situation when output from (or information about the result of) an event or phenomenon in the past will influence an occurrence or...
, such that each gene represses the next gene in the loop, and is repressed by the previous gene. In addition, green fluorescent protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
is used as a reporter so that the behavior of the network can be observed using fluorescence microscopy.
This analysis motivated two design features which were engineered into the genes:
First, to decrease leakiness the promoter regions were replaced with a tighter hybrid promoter which combined the λ PL promoter with LacL and TetR operator sequences.
Second, to reduce the disparity between the lifetimes of the repressor proteins and the mRNAs, a carboxy terminal tag based on the ssRA RNA sequence was added at the 3' end of each repressor gene. This tag is recognized by proteases which target the protein for degradation.
Implementation
The design was implemented using a low copy plasmidPlasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
encoding the repressilator, and the higher copy reporter, which were used to transform a culture of Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
.
Observations
To study the behavior of the repressilator single cells were isolated and the resulting colonies were observed over time using fluorescence microscopy. Observations were limited by the fact that the colonies entered a stationary phase after about 10 hours and approximately 5 oscillations.Conclusion
The repressilator is a milestone of synthetic biologySynthetic biology
Synthetic biology is a new area of biological research that combines science and engineering. It encompasses a variety of different approaches, methodologies, and disciplines with a variety of definitions...
which shows that genetic regulatory networks which perform a novel desired function can be designed and implemented. Further, this experiment gives new appreciation to the circadian clock found in many organisms, as they perform much more robustly than the repressilator.
External links
- Direct link to Hodgkin-Huxley model and a Description in BioModels DatabaseBioModels DatabaseBioModels Database is a free and open-source database for storing, exchanging and retrieving published quantitative models of biological interest...