CH391L/S12/Toggle Switches, Repressilators, and Counters

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===pIKE Toggle Switch Plasmid===
===pIKE Toggle Switch Plasmid===
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[[Image:Toggle Switch Threshold.jpg|thumb|right| Toggle Switch Threshold<cite>Gardner2000</cite>]]
The pIKE plasmid toggle switch differs from the pTAK plasmid by the P1 and R1 genes. In pIKE, P1 is the PLtetO-1 promoter and R1 is '‘tetR’'. This toggle switch is flipped by IPTG or aTc pulses.
The pIKE plasmid toggle switch differs from the pTAK plasmid by the P1 and R1 genes. In pIKE, P1 is the PLtetO-1 promoter and R1 is '‘tetR’'. This toggle switch is flipped by IPTG or aTc pulses.

Revision as of 02:07, 2 April 2012


Contents

Toggle Switches, Repressilators, and Counters

Toggle Switches

Toggle Switch Network[1]
Toggle Switch Network[1]

A toggle switch is a synthetic gene regulatory network which confers bistability. Bistability is where a system is under one of two possible conditions and never in between the two. To do so the cell has a threshold at which it switches between the two, so noise does not result in random flipping between the two states. Toggle switches consist of two promoters each of which drives expression of the repressor of the other. To switch between the two states, the inducer of the promoter currently being repressed is introduced long enough to cause the promoter’s expression to repress the originally active promoter. Gardner et al designed two toggle switch plasmids described below.[1]

pTAK Toggle Switch Plasmid

In the pTAK plasmid, the toggle switch consist of the Ptrc-2 promoter which is repressed by ‘‘lacI’’ and drives the expression of the temperature sensitive λ repressor (R1). R1 represses the second promoter in the switch, PLs1con (P1), which in turn drives the expression of ‘‘lacI’’.

Introduction of an IPTG or thermal pulse switches this toggle switch between its two states. The ‘‘gfpmut3’’ gene is located downstream of the Ptrc-2 promoter and is used to indicate what state the toggle switch is in as it only expresses fluorescence when the Ptrc-2 promoter is induced. If the P1 promoter is induced, then the Ptrc-2 promoter is prepressed and there is no fluorescence; this is called the "low state".[1]

pTAK and pIKE Plasmids[1]
pTAK and pIKE Plasmids[1]

pIKE Toggle Switch Plasmid

Toggle Switch Threshold[1]
Toggle Switch Threshold[1]

The pIKE plasmid toggle switch differs from the pTAK plasmid by the P1 and R1 genes. In pIKE, P1 is the PLtetO-1 promoter and R1 is '‘tetR’'. This toggle switch is flipped by IPTG or aTc pulses.

Gardner et al designed pIKE and pTAK with different ribosome binding sites to determine bistability under different conditions, and all but one pIKE plasmid conferred bistability which is possibly due to the fact that ‘'tetR’’ has less efficiency than the pTAK λ repressor. To test the bistability, the plasmids were induced with IPTG for 6 hours to express fluorescence, called the high state, and then grown 5 hours without IPTG. Plasmids that remained in the high state display bistability and ones that return to low states display monostability. Afterwards, the plasmids were treated with heat or aTC as appropriate for 7 hours to turn off GFP expression then removed for 5.5 hours; plasmids that remained in low state are considered bistable. [1]

The 2011 Duke iGEM team used zinc finger nucleases to modify genetic toggle switches in their iGEM project.

Repressilators

A repressilator is a synthetic gene network that uses the repression of genes in a negative feedback loop to create an oscillating network measured by GFP expression. This network involves three genes, each of which promote the expression of the repressor of the next gene.

Elowitz and Leibler designed a repressilator with ‘‘lacI’’as the first repressor. ‘‘LacI’’ represses the expression of the next repressor ‘‘tetR’’ which in turn represses the expression of the third repressor ‘‘cI’’. The ‘‘cI’’ repressor then represses the expression of ‘‘lacI’’. These three repressor genes along with their promoters were inserted into a low copy plasmid, and a reporter gene, GFP, was inserted into a high copy plasmid. Both plasmids were then cloned into ‘‘E. coli’’ cells grown in media containing IPTG. The cells were then transferred into media without IPTG and as they were transferred, each cell displayed a single oscillation of fluorescence.

In order to have proper temporal oscillation display rather than a single fixed state of transcription of the repressors, the repressors need to be strong, ribosome binding needs to be efficient, and the mRNA and protein decay rates of each gene need to be similar.

Elowitz and Leibler’s experiment is significant in the fact that it shows the ability to construct functional synthetic networks from common genes. Also repressilators have been likened to circadian clocks in organisms like cyanobacteria which oscillate in 24 hour patterns due to environmental change between night and day. The circadian oscillators are much more precise and efficient, however, which could be accounted for by the fact that they use both positive and negative feedback.[2]

The 2010 USTC iGEM team created a model to stimulate a repressilator as part of their project.

Counters

Synthetic cellular counters count events by expressing a reporter gene, mainly GFP, only after a certain number of pulses of an inducer. Counters are found naturally in systems such as telomere lengthening, and can be applied to tightly control processes like cell growth. Friedland et al constructed two types of synthetic genetic counters that can count up to three. [3]

Riboregulated Transcriptional Cascade

The riboregulated transcriptional cascade (RTC) consists of two promoters each of which is induced by arabinose, and the first promoter expresses a gene that promotes the expression of the second promoter which drives GFP expression. In a two-counter system, the first pulse of arabinose shortly induces the first promoter which encodes for T7 RNAP. The arabinose is then removed and the mRNA metabolized, and whatever small amount of T7 RNAP that was translated transcribes the second promoter to produce little amounts of GFP. Only at the second pulse does GFP expression increase significantly. In the three counter system the same method applies to a set of three promoters: T7 RNAP expression drives T3 RNAP expression which then drives GFP expression.

RTC synthetic gene counters can possibly be used to program cell death after a set amount of cell divisions; this can be very useful in containment of bioengineered cell strains. [3]

DNA Invertase Cascade

The DNA invertase cascade (DIC) system uses a single invertase memory module (SIMM) to count. An SIMM refers to a set of genes located between forward and reverse recombinase recognition sites. These genes include, in order, and inverted promoter, a recombinase gene, an ssrA tag for protein degredation, and a transcriptional terminator. An upstream promoter of the recombinase gene is turned on by a pulse of its inducer, usually arabinose; this promotes the expression of the recombinase which inverts the entire DNA region between the forward and reverse recombinase recognition sites. Once the SIMM is inverted, the upstream promoter can no longer promote the recombinase expression, and the inverted promoter is now in the right orientation to promote the next SIMM in the cascade at the next arabinose pulse. The number of SIMMs in the cascade determines if the system is a two-counter or three-counter. The last pulse in the cascade promotes GFP expression.

A multiple inducer DIC was also designed in which the three arabinose promoters are replaced with three different promoters such as one induced by aTc, one induced by arabinose, and the third induced by IPTG. High GFP expression is only seen when the three inducers are pulsed in that order. This allows a circuit to respond to a chosen sequence of events. [3]

The ETH Zurich iGEM team created a counter using toggle switches as part of their project.

References

  1. Gardner TS, Cantor CR, and Collins JJ. . pmid:10659857. PubMed HubMed [Gardner2000]
  2. Elowitz MB and Leibler S. . pmid:10659856. PubMed HubMed [Elowitz2000]
  3. Friedland AE, Lu TK, Wang X, Shi D, Church G, and Collins JJ. . pmid:19478183. PubMed HubMed [Friedland2009]
  4. Smolke CD. . pmid:19478174. PubMed HubMed [Smolke2009]
All Medline abstracts: PubMed HubMed
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