CH391L/S12/Toggle Switches, Repressilators, and Counters - Revision history

Etienne Robillard: /* Introduction */

Etienne Robillard — Sat, 15 Dec 2012 00:02:24 GMT

Introduction

←Older revision		Revision as of 00:02, 15 December 2012
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	==Introduction==		==Introduction==

-	Toggle switches, repressilators, and counters are synthetic biological information processing systems to control gene expression based on environmental cues.<cite>Smolke2009</cite> Counters use memory and time delay to process the frequency of an event which has applications in recording environmental conditions. Toggle switches use memory by switching into one fixed state following its induction signal which could be applied to detecting pollutant levels in the environment. The toggle switch could keep memory of a certain level of a pollutant and display a reporter gene as a signal.<cite>Collins2012</cite> For example, June Medford from Colorado State University has engineered toggle switches in plants that turn off ~~clorophyll~~ production and turn white when they detect explosive chemicals.	+	Toggle switches, repressilators, and counters are synthetic biological information processing systems to control gene expression based on environmental cues.<cite>Smolke2009</cite> Counters use memory and time delay to process the frequency of an event which has applications in recording environmental conditions. Toggle switches use memory by switching into one fixed state following its induction signal which could be applied to detecting pollutant levels in the environment. The toggle switch could keep memory of a certain level of a pollutant and display a reporter gene as a signal.<cite>Collins2012</cite> For example, June Medford from Colorado State University has engineered toggle switches in plants that turn off chlorophyll production and turn white when they detect explosive chemicals.

	==Toggle Switches==		==Toggle Switches==

Etienne Robillard: /* Counters */

Etienne Robillard — Fri, 14 Dec 2012 23:53:40 GMT

Counters

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	[[Image:RTC.jpg\|thumb\|right\| RTC Network<cite>Friedland2009</cite>]]		[[Image:RTC.jpg\|thumb\|right\| RTC Network<cite>Friedland2009</cite>]]

-	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.<cite>Friedland2009</cite> 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.	+	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.<cite>Friedland2009</cite> 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 counters 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.		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.

Jeffrey E. Barrick: /* pTAK Toggle Switch Plasmid */

Jeffrey E. Barrick — Sun, 29 Apr 2012 17:52:05 GMT

pTAK Toggle Switch Plasmid

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	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).<cite>Gardner2000</cite> R1 represses the second promoter in the switch, PLs1con (P1), which in turn drives the expression of ''lacI''.		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).<cite>Gardner2000</cite> 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".	+	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 repressed and there is no fluorescence; this is called the "low state".

	===pIKE Toggle Switch Plasmid===		===pIKE Toggle Switch Plasmid===

Razan Alnahhas: /* Introduction */

Razan Alnahhas — Mon, 09 Apr 2012 00:46:55 GMT

Introduction

←Older revision		Revision as of 00:46, 9 April 2012
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	==Introduction==		==Introduction==

-	Toggle switches, repressilators, and counters are synthetic biological information processing systems to control gene expression based on environmental cues.<cite>Smolke2009</cite> Counters use memory and time delay to process the frequency of an event which has applications in recording environmental conditions. Toggle switches use memory by switching into one fixed state following its induction signal which could be applied to detecting pollutant levels in the environment. The toggle switch could keep memory of a certain level of a pollutant and display a reporter gene as a signal.<Collins2012</cite> For example, June Medford from Colorado State University has engineered toggle switches in plants that turn off clorophyll production and turn white when they detect explosive chemicals.	+	Toggle switches, repressilators, and counters are synthetic biological information processing systems to control gene expression based on environmental cues.<cite>Smolke2009</cite> Counters use memory and time delay to process the frequency of an event which has applications in recording environmental conditions. Toggle switches use memory by switching into one fixed state following its induction signal which could be applied to detecting pollutant levels in the environment. The toggle switch could keep memory of a certain level of a pollutant and display a reporter gene as a signal.<cite>Collins2012</cite> For example, June Medford from Colorado State University has engineered toggle switches in plants that turn off clorophyll production and turn white when they detect explosive chemicals.

	==Toggle Switches==		==Toggle Switches==

Razan Alnahhas: /* References */

Razan Alnahhas — Mon, 09 Apr 2012 00:45:48 GMT

References

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	#Smolke2009 pmid=19478174		#Smolke2009 pmid=19478174

-	#~~Collins 2012~~ pmid=22378128	+	#Collins2012 pmid=22378128

	#Gardner2000 pmid=10659857		#Gardner2000 pmid=10659857

Razan Alnahhas: /* Toggle Switches, Repressilators, and Counters */

Razan Alnahhas — Mon, 09 Apr 2012 00:00:10 GMT

Toggle Switches, Repressilators, and Counters

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	=Toggle Switches, Repressilators, and Counters=		=Toggle Switches, Repressilators, and Counters=
		+
		+	==Introduction==
		+
		+	Toggle switches, repressilators, and counters are synthetic biological information processing systems to control gene expression based on environmental cues.<cite>Smolke2009</cite> Counters use memory and time delay to process the frequency of an event which has applications in recording environmental conditions. Toggle switches use memory by switching into one fixed state following its induction signal which could be applied to detecting pollutant levels in the environment. The toggle switch could keep memory of a certain level of a pollutant and display a reporter gene as a signal.<Collins2012</cite> For example, June Medford from Colorado State University has engineered toggle switches in plants that turn off clorophyll production and turn white when they detect explosive chemicals.

	==Toggle Switches==		==Toggle Switches==
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	==References==		==References==
	<biblio>		<biblio>
		+
		+	#Smolke2009 pmid=19478174
		+
		+	#Collins 2012 pmid=22378128

	#Gardner2000 pmid=10659857		#Gardner2000 pmid=10659857
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	#Friedland2009 pmid=19478183		#Friedland2009 pmid=19478183
-
-	~~#Smolke2009 pmid=19478174~~

	</biblio>		</biblio>

Jeffrey E. Barrick: /* DNA Invertase Cascade */

Jeffrey E. Barrick — Fri, 06 Apr 2012 16:54:53 GMT

DNA Invertase Cascade

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	[[Image:DIC Network.jpg\|thumb\|left\|DIC Network<cite>Friedland2009</cite>]]		[[Image:DIC Network.jpg\|thumb\|left\|DIC Network<cite>Friedland2009</cite>]]

-	The DNA invertase cascade (DIC) system uses a single invertase memory module (SIMM) to count.<cite>Friedland2009</cite> 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.	+	The DNA invertase cascade (DIC) system uses a single invertase memory module (SIMM) to count.<cite>Friedland2009</cite> 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 degradation, 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.

	[[Image:DIC Multiple Inducer.jpg\|thumb\|right\| DIC Multiple Inducer Network<cite>Friedland2009</cite>]]		[[Image:DIC Multiple Inducer.jpg\|thumb\|right\| DIC Multiple Inducer Network<cite>Friedland2009</cite>]]

Jeffrey E. Barrick: /* Repressilators */

Jeffrey E. Barrick — Fri, 06 Apr 2012 16:20:31 GMT

Repressilators

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	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.		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.

-	[http://2010.igem.org/Team:USTC_Software/Repressilator The 2010 USTC iGEM team] created a model to ~~stimulate~~ a repressilator as part of their project.	+	[http://2010.igem.org/Team:USTC_Software/Repressilator The 2010 USTC iGEM team] created a model to simulate a repressilator as part of their project.

	==Counters==		==Counters==

Brian Renda at 20:15, 2 April 2012

Brian Renda — Mon, 02 Apr 2012 20:15:48 GMT

←Older revision		Revision as of 20:15, 2 April 2012
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	==Repressilators==		==Repressilators==

-	[[Image:Repressilator Network.jpg\|thumb\|left\| Repressilator Network<cite>~~Elowtiz2000~~</cite>]]	+	[[Image:Repressilator Network.jpg\|thumb\|left\| Repressilator Network<cite>Elowitz2000</cite>]]

	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.<cite>Elowitz2000</cite> This network involves three genes, each of which promote the expression of the repressor of the next gene.		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.<cite>Elowitz2000</cite> This network involves three genes, each of which promote the expression of the repressor of the next gene.

Razan Alnahhas: /* pIKE Toggle Switch Plasmid */

Razan Alnahhas — Mon, 02 Apr 2012 17:19:00 GMT

pIKE Toggle Switch Plasmid

←Older revision		Revision as of 17:19, 2 April 2012
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	The pIKE plasmid toggle switch differs from the pTAK plasmid by the P1 and R1 genes.<cite>Gardner2000</cite> 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.<cite>Gardner2000</cite> 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.	+	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.

	[http://2011.igem.org/Team:Duke/Project The 2011 Duke iGEM team] used zinc finger nucleases to modify genetic toggle switches in their iGEM project.		[http://2011.igem.org/Team:Duke/Project The 2011 Duke iGEM team] used zinc finger nucleases to modify genetic toggle switches in their iGEM project.