a central focus of synthetic biology is constructing isolated gene regulatory networks in living cells in order to determine their dynamical behavior in a given environment . the complexity of naturally occurring gene networks makes this determination difficult , and many investigators have concentrated on smaller artificial circuits ' in an attempt to understand fundamental principles ( hasty et al , 2002 ; sprinzak and elowitz , 2005 ) . these smaller networks are more amenable to computational modeling , construction and modification . in addition , the systematic construction of genetic circuits from a set of quantitative design principles will accelerate progress towards therapeutic applications . many cellular functions rely on underlying regulatory processes that are highly dynamic . in the context of interconnected gene regulatory and signalling networks , the persistence of mrna and protein molecules has a large effect on the overall behavior of the cell . this effect often manifests in the underlying network dynamics , where degradation rates can play a central role in mitigating crucial timing events . naturally occurring components these components are usually stable , and this stability constrains the ability to monitor highly dynamic circuits and to construct circuits that possess desired response characteristics . for example , gfp is often used as a reporter in native and artificial synthetic genetic networks , due to its ability to be expressed in various hosts without interfering with cellular function . however , gfp is stable in most cells on a time scale of hours to days ( andersen et al , 1998 ) . once stable gfp is expressed , it is cleared from the system only through growth and subsequent dilution , making measurements at higher temporal resolution difficult to interpret . however , such an increase in temporal responsiveness is not without cost , since it reduces detectable signal . ideally , one could control the stability of a network component , tuning it to balance the needs of signal detection and dynamical resolution . there have been several studies that use native protein degradation systems to make network components less persistent . andersen et al ( 1998 ) used the ssra system in escherichia coli to target proteins to degradation pathways . in bacteria , polypeptides that stall during translation ( e.g. , under starvation conditions ) have an 11-amino - acid tag added to their c - terminus by a small ssra molecule . this tag is specifically recognized by the clpxp proteasome , and tagged proteins are degraded ( gottesman et al , 1998 ; karzai et al , 2000 ) . more recently , it was demonstrated that degradation in e. coli could be increased with a modified tag that binds a helper molecule ( mcginness et al , 2006 ) . in saccharomyces cerevisiae , a similar system was described that made translational fusions between a given protein and a domain of cln2 , a yeast protein that is degraded quickly ( mateus and avery , 2000 ) . it has also been shown that modification of the s. cerevisiae n - degron signal sequence can lead to impressive destabilization of reporters down to a half - life of 2 min ( hackett et al , 2006 ) . while all of these systems produce more dynamic proteins , none focused on developing tunability over a wide range of degradation rates . in addition , the overexpression of native degradation components in the same organism for the purpose of increasing degradation can have undesired pleiotropic effects . we have constructed a s. cerevisiae strain ( cgd699 ) that allows tunable degradation of a tagged protein . to accomplish this , we expressed a modified e. coli clpxp protease in yeast under the control of a repressible promoter . proteins that are tagged with the ssra tag are quickly degraded , and this degradation rate is controlled by the induction level of clpxp . we integrated the two e. coli genes ( clpx and clpp ) that code for the clpxp protease into the yeast genome ( figure 1 , and materials and methods ) . we found that the clpx gene needed to be modified with 10 silent mutations to be expressed in yeast ( see materials and methods ) . these two genes were placed under the control of two separate copies of the modified adh1i promoter ( blake , 2003 ) , at two different loci in the yeast genome . additionally , we integrated mlaci , a mammalian - enhanced version of laci ( cronin et al , 2001 ) , controlled by a wild - type adh1 promoter . the wild - type version of the adh1 promoter exhibits constitutive expression , while the adh1i promoter is repressed by laci in the absence of iptg . addition of iptg to the medium results in clpxp production and degradation of a tagged protein . to demonstrate the utility of this approach , we also integrated a yegfp gene tagged with an 11-amino - acid ssra tag ( aandenyalaa ) , under the control of the gal1 promoter into cgd699 . this promoter is fully induced by 0.5% w / v galactose and repressed by 2% w / v glucose . cgd699 cells grown in the presence of galactose produce gfp and are fluorescent , and gfp production ceases if the carbon source in the media is switched from galactose to glucose . observations of growth rate and morphology indicate that the exogenous proteases cause no deleterious cellular effects over a wide range of iptg levels ( figure 2a and b ) . while coexpression of tagged yegfp and the degradation machinery resulted in almost complete loss of fluorescence , coexpression of untagged yegfp with the degradation machinery showed no significant drop in fluorescence ( figure 2c ) . we further investigated the ability of cgd699 cells to degrade tagged yegfp in response to iptg induction by microscopy in a microfluidic chamber . prior to imaging , the cells were grown in log phase for 2436 h in media containing 0.5% galactose and the experimental iptg concentration . these cells were then loaded into a yeast microfluidic chamber ( cookson et al , 2005 ) that was designed to allow temporal switching of inducer ( bennett et al , 2007 ) . the growth media was switched to media containing iptg and 2% glucose to shut off gfp production from the gal1 promoter . figure 3a shows a series of bright - field and corresponding fluorescent images for cells exhibiting active degradation in the 1 mm iptg condition ( also see supplemental movie ) . in cells with yegfp - ssra but without clpxp incorporation , the half - life of gfp was 10419 min . this half - life is the result of growth - related dilution ( note that the doubling time of the batch culture grown on galactose was approximately 125 min ; see figure 2a ) . the induction of varied expression of the clpxp protease allows the reduction of the half - life of gfp to as low as 22 min at 5 mm iptg . previous studies have used exponential fits to characterize the half - life of the reporter . we found that modeling decay as arising from a set of enzymatic michaelis however , in order to systematically compare with the previous fluorescent reporter degradation studies , we chose to first analyze the fluorescence trajectories with exponential fits that were reasonably accurate ( figure 3b ) . from these fits , we were able to calculate the mean half - life for each concentration of iptg , as shown in figure 3c . the half - life decreases from a value of 91 min for no iptg to 22 min for media containing 5 mm iptg . in addition to the exponential fitting , we used the experimental data to determine how the degradation process should be modeled . for this , we assumed that both the gal1 and adh1 promoters are constitutive , and that yegfp - ssra , clpxp and mlaci have reached equilibrium before the introduction of glucose . in this case , the transcription of the gal1 promoter stops once glucose has been added and its mrna begins to decay exponentially from its steady - state value . however , the translation of the mrna continues , leading to an ode describing the dynamics of the concentration of yegfp - ssra : where x is the concentration of yegfp - ssra ,  is the degradation rate of the gal1 mrna , c is a constant related to the transcription and translation rates of gal1 ,  is the dilution rate and v and k are the michaelis we assume that glucose is added at t=0 , so that is the steady - state initial concentration . because the constant v is proportional to the concentration of the enzyme ( clpxp ) , it will increase with increasing levels of iptg ( which activates clpxp production ) . therefore , increasing the concentration of iptg will increase the degradation rate of the substrate ( yegfp - ssra ) . figure 3d shows a comparison between single - cell data from three trials ( symbols ) and time series obtained from a numerical best - fit algorithm of equation 1 ( solid lines ) . the excellent agreement implies that clpxp - mediated degradation is a michaelis  menten enzymatic process , as opposed to first - order decay , which is often assumed in models describing signalling or gene - regulatory networks . we have adapted the prokaryotic ssra tagging system ( andersen et al , 1998 ; gottesman et al , 1998 ) for use in eukaryotic yeast cells . the cgd699 strain allows for the tunable degradation of any protein by adding a short amino - acid tag . in reporter systems , the competing requirements of signal detection and dynamical resolution the small size of the tag simplifies construction of tagged genes by pcr amplification or use of a tagging vector , and many genes can be tagged in parallel . several variant ssra tags have been described ( andersen et al , 1998 ; mcginness et al , 2006 ) , allowing another level of control . induction of clpxp was non - toxic , as we found no significant change in growth rate or morphology at different iptg concentrations . in addition , although a yeast homologue to clpx has been identified ( van dyck et al , 1998 ) , it is located in the mitochondria and acts as a chaperone and not a protease . the degradation components are stably integrated into the yeast genome , which allows it to be adapted for use with any number of tagged targets , either integrated or expressed from a plasmid . due to the simple nature of the system , it might be portable into other genetically tractable eukaryotic models . there is evidence that bacterial clpx and clpp can interact with mammalian mitochondrial clpx and clpp ( kang et al , 2002 ) , but the restricted subcellular localization of these mammalian proteins should limit in vivo interaction . increased degradation might be achievable by coexpressing the e. coli sspb gene , which facilitates ssra tag binding to the clpxp protease ( hersch et al , 2004 ) . although we have only used the cgd699 strain with a fluorescent reporter protein , it should be applicable to any protein that can support the short c - terminal ssra ( huh et al , 2003 ) . we expect that this system will prove useful for a variety of applications where increased temporal resolution is desired , both as a tool in the construction of small - scale synthetic networks ( hasty et al , 2002 ; sprinzak and elowitz , 2005 ) , and in systems biology studies , which seek to unravel network connectivity through perturbation of a given component ( ideker et al , 2001 ; faith et al , 2007 ) . s. cerevisiae cells were grown at 30c with 300 r.p.m . shaking in sd dropout media containing appropriate amino - acid supplements . this was supplemented with 20 mg / l tryptophan , histidine and uracil and 100 mg / l leucine , as appropriate . e. coli dh5 cells were grown at 37c with shaking at 300 r.p.m . in lb medium supplemented with 100 g / ml ampicillin to maintain plasmids . dna manipulations were carried out by standard techniques ( sambrook and russell , 2001 ) . yegfp ( sheff and thorn , 2004 ) was cloned from prs31-yg ( volfson et al , 2006 ) into an ssra tagging vector that attached an ssra tag ( aandenyalaa ) to the c - terminal end , and then cloned back into prs31-yg and integrated into the his3 locus of s. cerevisiae strain k699 . clpx and clpp were cloned from e. coli 2.300 by genomic pcr using phusion polymerase ( new england biolabs ) . clpp was placed behind the laci - repressible adh1i promoter on prs3gfpi ( blake , 2003 ) , replacing the gfp gene . terminator cassette was then cloned into prs404 and incorporated into the s. cerevisiae trp1 locus . yeast - optimized clpx was incorporated into the s. cerevisiae ura3 locus via prs406 using a similar procedure . mammalian - enhanced laci ( cronin et al , 2001 ) was placed under the wild - type adh1 promoter on prs3gfpa , replacing the gfp gene . terminator cassette was then cloned into prs405 and incorporated into the s. cerevisiae leu2 locus . our initial attempts to construct this strain resulted in fluorescent signal but no corresponding drop in fluorescence when clpx and clpp were induced . pcr results showed that the mrna was transcribed , but western blotting results showed that the clpx was not translated ( data not shown ) . we suspected that codon bias between the two organisms was a factor , and optimized clpx for yeast expression . sources of possibly deleterious codon bias were identified by comparing codon usages between e. coli and s. cerevisiae using the codon usage database ( http://www.kazusa.or.jp/codon/ ) . we introduced 10 silent mutations in two rounds of multiple mutagenesis using a previously described method ( sawano and miyawaki , 2000 ) , and using phusion polymerase ( neb ) . we omitted the megaprimer step , instead transforming single - stranded dna directly into ultracompetent xl-10 gold cells ( stratagene ) . the following primers were used ( mutations induced are set bold ) : primer 1 : ggtcgcggtatacaaccattacaaaagattgcgcaacggcgataccagcaatggcgtcgag primer 2 : gctgctggctgaaacgctggctagattgctggatgttccgttcaccatggccgac primer 3 : ctatcgctaagaaagcgatggctagaaaaaccggtgcccgtggcct . a microfabricated chemostatic growth chamber that contained an on - chip media switch was used to measure clpxp - mediated gfp decay within single cells . the microdevices were fabricated using well - documented poly - dimethylsiloxane ( pdms , dow corning sylgard 184 ) replica molding techniques ( xia and whitesides , 1998 ) . chambers were fabricated to 4 m in depth to facilitate quantitative fluorescence imaging of single cells ( cookson et al , 2005 ) . before experimentation , cells were grown from plates in appropriate auxotrophic media containing 0.5% galactose , 2% raffinose and the experimental iptg condition for at least 24 h in batch culture . yeast cells were loaded into microfabricated growth chambers at an od600 of 0.40.6 and allowed to grow for 1 h in loading media . they were then transferred to media containing 2% ( w / v ) glucose and no galactose , the experimental iptg concentration and a red fluorescent tracer dye ( sulforhodamine 101 , sigma ) . cells were imaged over the course of 412 h in bright field , green fluorescence and red fluorescence at 5 min intervals . mean pixel intensity within each object was calculated and tracked through each time series using object centroids . temporal profiles of 1530 cells that exhibited decay responses for each iptg concentration were collected . all image processing , segmentation and tracking procedures were performed using software developed in matlab ( the mathworks inc . ) . s. cerevisiae cells were grown at 30c with 300 r.p.m . shaking in sd dropout media containing appropriate amino - acid supplements . this was supplemented with 20 mg / l tryptophan , histidine and uracil and 100 mg / l leucine , as appropriate . e. coli dh5 cells were grown at 37c with shaking at 300 r.p.m . in lb medium supplemented with 100 g / ml ampicillin to maintain plasmids . dna manipulations were carried out by standard techniques ( sambrook and russell , 2001 ) . yegfp ( sheff and thorn , 2004 ) was cloned from prs31-yg ( volfson et al , 2006 ) into an ssra tagging vector that attached an ssra tag ( aandenyalaa ) to the c - terminal end , and then cloned back into prs31-yg and integrated into the his3 locus of s. cerevisiae strain k699 . clpx and clpp were cloned from e. coli 2.300 by genomic pcr using phusion polymerase ( new england biolabs ) . clpp was placed behind the laci - repressible adh1i promoter on prs3gfpi ( blake , 2003 ) , replacing the gfp gene . terminator cassette was then cloned into prs404 and incorporated into the s. cerevisiae trp1 locus . yeast - optimized clpx was incorporated into the s. cerevisiae ura3 locus via prs406 using a similar procedure . mammalian - enhanced laci ( cronin et al , 2001 ) was placed under the wild - type adh1 promoter on prs3gfpa , replacing the gfp gene . terminator cassette was then cloned into prs405 and incorporated into the s. cerevisiae leu2 locus . our initial attempts to construct this strain resulted in fluorescent signal but no corresponding drop in fluorescence when clpx and clpp were induced . pcr results showed that the mrna was transcribed , but western blotting results showed that the clpx was not translated ( data not shown ) . we suspected that codon bias between the two organisms was a factor , and optimized clpx for yeast expression . sources of possibly deleterious codon bias were identified by comparing codon usages between e. coli and s. cerevisiae using the codon usage database ( http://www.kazusa.or.jp/codon/ ) . we introduced 10 silent mutations in two rounds of multiple mutagenesis using a previously described method ( sawano and miyawaki , 2000 ) , and using phusion polymerase ( neb ) . we omitted the megaprimer step , instead transforming single - stranded dna directly into ultracompetent xl-10 gold cells ( stratagene ) . the following primers were used ( mutations induced are set bold ) : primer 1 : ggtcgcggtatacaaccattacaaaagattgcgcaacggcgataccagcaatggcgtcgag primer 2 : gctgctggctgaaacgctggctagattgctggatgttccgttcaccatggccgac primer 3 : ctatcgctaagaaagcgatggctagaaaaaccggtgcccgtggcct . a microfabricated chemostatic growth chamber that contained an on - chip media switch was used to measure clpxp - mediated gfp decay within single cells . the microdevices were fabricated using well - documented poly - dimethylsiloxane ( pdms , dow corning sylgard 184 ) replica molding techniques ( xia and whitesides , 1998 ) . chambers were fabricated to 4 m in depth to facilitate quantitative fluorescence imaging of single cells ( cookson et al , 2005 ) . before experimentation , cells were grown from plates in appropriate auxotrophic media containing 0.5% galactose , 2% raffinose and the experimental iptg condition for at least 24 h in batch culture . yeast cells were loaded into microfabricated growth chambers at an od600 of 0.40.6 and allowed to grow for 1 h in loading media . they were then transferred to media containing 2% ( w / v ) glucose and no galactose , the experimental iptg concentration and a red fluorescent tracer dye ( sulforhodamine 101 , sigma ) . cells were imaged over the course of 412 h in bright field , green fluorescence and red fluorescence at 5 min intervals . mean pixel intensity within each object was calculated and tracked through each time series using object centroids . temporal profiles of 1530 cells that exhibited decay responses for each iptg concentration were collected . all image processing , segmentation and tracking procedures were performed using software developed in matlab ( the mathworks inc . ) .
protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators . 
 turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses . here 
 , we use components of the escherichia coli degradation machinery to construct a saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein . using a microfluidic platform tailored for single - cell fluorescence measurements , 
 we monitor protein decay rates after repression using an ssra - tagged fluorescent reporter . 
 we observe a half - life ranging from 91 to 22 min , depending on the level of activation of the degradation genes . 
 computational modeling of the underlying set of enzymatic reactions leads to gfp decay curves that are in excellent agreement with the observations , implying that degradation is governed by michaelis  menten - type interactions . 
 in addition to providing a reporter with tunable dynamic resolution , our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways .