Repressilator

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The repressilator is a novel synthetic genetic regulatory network reported in a paper^[1] by Michael B. 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 a clock but resembles no known natural clock. 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.

1 Design
2 Implementation
3 Observations
4 Conclusion
5 References

[edit] Design

The repressilator consists of three genes connected in a feedback loop, 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 repressilator genetic regulatory network.

The design of the repressilator was guided by two simple mathematical models, one continuous and deterministic and the other discrete and stochastic.

A discrete and stochastic simulation of the repressilator.

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 P_L 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.

[edit] Implementation

The design was implemented using a low copy plasmid encoding the repressilator, and the higher copy reporter, which were used to transform a culture of Escherichia coli.

The plasmids used to implement the repressilator in Escherichia coli.

[edit] 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.

The fluorescent and bright field images of a single transformed cell over a period of 10 hours.

Observations of various experiments. Examples a-d show the variability of in the period and amplitude of oscillations. Examples e and f are negative controls. Example e has been disrupted, and example f contains only the reporter plasmid.

[edit] Conclusion

The repressilator is a milestone of synthetic biology which shows that we can design and implement genetic regulatory networks which perform a novel desired function. Further, this experiment gives new appreciation to the circadian clocks found in many organisms, as they perform much more robustly than the repressilator.