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In class first assignment.  Cloned wiki.  Feedback on discussion page.
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Drew Tack's first assignment.  Cloned wiki from spring 2012 semester.  Feedback on discussion page.
  
Bacterial communities are capable of producing a wide variety of odor molecules. At its most basic, an odorant is simply a volatile organic compound (VOC) capable of triggering an olfactory response. These compounds fall into many categories, and a few examples will be discussed here. These compounds can be produced for a variety of reasons, from simple metabolic byproducts to targeted cellular messengers. Although bacteria don't have an olfactory system in the same sense as higher organisms, there is evidence that bacterial communities can communicate via volatile airborne compounds and that they may be an important bacterial defense sensor.
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[[Category:CH391L_S12]]
  
==Natural Bacterial Odorant Production==
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=Toggle Switches, Repressilators, and Counters=
  
It's difficult not to view bacterial odorant production through a human lens, but it's important to remember that the particular descriptions of these compounds are purely coincidental when it comes to smell. When it comes to odorants produced naturally by bacterial populations, it's much more appropriate to view the compounds as products of synthetic pathways and not clouding them with human "uses" or "what they remind us of". That being said, naturally occurring bacterial odorants have been used by humans and other organisms to very interesting ends.
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==Introduction==
  
Carnivorous mammals including dogs, wolves and hyenas possess potent scent glands near their anus called apocrine glands. These sacs produce a thick liquid or paste that the animals use to mark territory and identify each other. This is why you see dogs sniffing near each other's anuses when meeting each other. Human and primate apocrine glands migrated upwards to our chest and armpit regions as we began to walk upright, and today they remain the source of our armpit odors. The glands produce a mostly odorless liquid, which is in turn metabolized by hundreds of species of skin-dwelling bacteria, with high densities of [http://en.wikipedia.org/wiki/Corynebacterium Corynebacteria]. Similar compounds are produced by digested secretions from the groin and foot regions.
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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.
  
Lipids in the apocrine and sebaceous secretions are digested by lipase enzymes into volatile compounds like butyric acid[[Image:Butyric-acid-3D-balls.png|thumb|right| Butyric acid]], also present in vomit, butter and Parmesan cheese and detectable by humans down to 10 ppm. There are hundreds such compounds produced by skin-dwelling bacterial species.
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==Toggle Switches==
  
In hyena populations, the particular makeup of apocrine microbial communities [http://scientistatwork.blogs.nytimes.com/2011/07/07/do-microbes-help-hyenas-communicate/# can differ between social groups and even sexes]. It is even thought that individuals that move between social groups must adopt the apocrine microbial community of their new pack to be accepted.
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[[Image:Toggle Switch.jpg|thumb|right| Toggle Switch Network<cite>Gardner2000</cite>]]
  
==Synthetic Bacterial Odorant Production==
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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.<cite>Gardner2000</cite> 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.
  
Several groups have engineered bacteria to produce unnatural odorant molecules. This method is similar to metabolic engineering pigment or other biosynthetic pathways in that exogenous single- or multi-enzymatic pathways are introduced to convert precursor molecules to a desired volatile compound.
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===pTAK Toggle Switch Plasmid===
  
The [http://openwetware.org/wiki/IGEM:MIT/2006 2006 MIT iGEM team] created an <i>E. coli</i> strain that emitted a wintergreen scent (methyl salicylate) in log phase growth and then switched to producing a banana scent (isoamyl acetate) when nutrients became scarce. They used a chassis strain called YYC912 (tnaA5-)  that is deficient in indole production (the characteristic foul-smelling compound of <i>E. coli</i> cultures). Genes were introduced that could synthesize the VOCs from media additives (salicylic acid) or natural amino acid production (leucine).
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[[Image:pTAK and pIKE.jpg|thumb|left| pTAK and pIKE Plasmids<cite>Gardner2000</cite>]]
  
[[Image:Eau_du_coli_parts.png]]
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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''.
  
They then assembled a circuit where the default state for M. salicylate production during exponential growth is "on". The wintergreen pathway is under control of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J45992 osmY stationary phase promoter] upstream of a [http://partsregistry.org/wiki/index.php/Part:BBa_Q04401 TetR transcriptional inverter]. The isoamyl acetate pathway is under control of the osmY promoter alone. When osmY transcription is activated at stationary phase, the wintergreen pathway is inverted to "off" and the banana pathway is activated.
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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".
  
[[Image:IGEM_MIT2006_Fullsystem.jpg]]
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===pIKE Toggle Switch Plasmid===
  
The Parts Registry contains pathways to produce many other VOCs including vanillin, pinene, jasmine and floral odors (among others).
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[[Image:Toggle Switch Threshold.jpg|thumb|right| Toggle Switch Threshold<cite>Gardner2000</cite>]]
  
==Bacterial "Olfaction"==
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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.
Until recently, the odorant molecules produced by bacteria were not thought to have much biochemical significance to the microbes themselves. Via a laboratory accident, <i>Bacillus licheniformis</i> was found to respond to the presence of gaseous ammonia by forming a biofilm<cite>Nijland2010</cite>. When the researchers were trying to save space on a plate by culturing two different strains together, those closest to wells producing ammonia turned red as biofilm production was induced.
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[[Image:Abstract.png]]
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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.  
  
Acetaldehyde detection and subsequent gene activation by <i>Aspergillus nidulans</i> has been characterized and engineered into mammalian cells in culture. Reporter genes or genes of interest can be put under control of the <i>A. nidulans</i> acetaldehyde-sensitive transactivator/promoter and turned on via acetaldehyde in a dose and concentration dependent manner<cite>Weber2004</cite>.  
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[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.
  
[[Image:F1.jpeg|thumb|left|Vanillin on/off system from Gutzinger et al. 2012]]
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==Repressilators==
  
As more odorant-sensitive pathways are discovered and characterized, olfactory circuit options for bioengineering will be expanded, as has recently been shown for a vanillin-inducible system in mammalian cells<cite>Gutzinger2012</cite>.
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[[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.
 +
 
 +
Elowitz and Leibler designed a repressilator with ''lacI'' as the first repressor.<cite>Elowitz2000</cite> ''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.
 +
 
 +
[[Image:Oscillation Image.jpg|thumb|right| GFP Oscillation<cite>Elowitz2000</cite>]]
 +
 
 +
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.
 +
 
 +
[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==
 +
 
 +
Synthetic cellular counters count events by expressing a reporter gene, mainly GFP, only after a certain number of pulses of an inducer.<cite>Friedland2009</cite> 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.
 +
 
 +
=== Riboregulated Transcriptional Cascade===
 +
 
 +
[[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 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.
 +
 
 +
===DNA Invertase Cascade===
 +
 
 +
[[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 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>]]
 +
 
 +
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.<cite>Friedland2009</cite> 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.
 +
 
 +
[http://parts.mit.edu/wiki/index.php/ETH_Zurich_2005 The ETH Zurich iGEM team] created a counter using toggle switches as part of their project.
  
 
==References==
 
==References==
 
<biblio>
 
<biblio>
#Nijland2010 pmid=20721987
 
//Bacterial olfaction
 
  
#Weber2004 pmid=15502819
+
#Smolke2009 pmid=19478174
//Acetaldehyde responsive gene expression in mammalian cells
+
 
 +
#Collins2012 pmid=22378128
 +
 
 +
#Gardner2000 pmid=10659857
 +
 
 +
#Elowitz2000 pmid=10659856
 +
 
 +
#Friedland2009 pmid=19478183
  
#Gutzinger2012 pmid=22187155
+
</biblio>
//Vanillin-induced gene expression in mammalian cells
+

Latest revision as of 19:00, 20 January 2014

Drew Tack's first assignment. Cloned wiki from spring 2012 semester. Feedback on discussion page.

Contents

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.[1] 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.[2] 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

File:Toggle Switch.jpg
Toggle Switch Network[3]

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.[3] 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.

pTAK Toggle Switch Plasmid

File:PTAK and pIKE.jpg
pTAK and pIKE Plasmids[3]

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).[3] 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 repressed and there is no fluorescence; this is called the "low state".

pIKE Toggle Switch Plasmid

File:Toggle Switch Threshold.jpg
Toggle Switch Threshold[3]

The pIKE plasmid toggle switch differs from the pTAK plasmid by the P1 and R1 genes.[3] 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.

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

Repressilators

File:Repressilator Network.jpg
Repressilator Network[4]

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.[4] 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.[4] 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.

File:Oscillation Image.jpg
GFP Oscillation[4]

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.

The 2010 USTC iGEM team created a model to simulate 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.[5] 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.

Riboregulated Transcriptional Cascade

RTC Network[5]

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.[5] 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.

DNA Invertase Cascade

File:DIC Network.jpg
DIC Network[5]

The DNA invertase cascade (DIC) system uses a single invertase memory module (SIMM) to count.[5] 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.

File:DIC Multiple Inducer.jpg
DIC Multiple Inducer Network[5]

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.[5] 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.

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

References

Error fetching PMID 19478174:
Error fetching PMID 22378128:
Error fetching PMID 10659857:
Error fetching PMID 10659856:
Error fetching PMID 19478183:
  1. Error fetching PMID 19478174: [Smolke2009]
  2. Error fetching PMID 22378128: [Collins2012]
  3. Error fetching PMID 10659857: [Gardner2000]
  4. Error fetching PMID 10659856: [Elowitz2000]
  5. Error fetching PMID 19478183: [Friedland2009]
All Medline abstracts: PubMed | HubMed