Improving the Lac System for Synthetic Biology

Penumetcha, Pallavi; Lau, Kin H.; Zhu, Xiao Fan; Davis, Kelly J.; Eckdahl, Todd T.; Campbell, A. Malcolm

doi:10.1893/011.081.0104

Cited by 22 publications

(19 citation statements)

References 13 publications

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“…This conclusion is valid (of course, under certain experimentally controllable conditions pointed out in this work) for all parameter values, and provides a strong theoretical guarantee missing from other synthetic biology designs. Some of conditions include: ( i ) a reduced promoter leakiness [ 51 ], and ( ii ) unsaturated levels of AAA+ proteases ClpXP.…”

Section: Resultsmentioning

confidence: 99%

“…Models always involve simplifications of reality. The impact of several such simplifications and assumptions, particularly those impacting monotonicity properties and bistabiliy regions, are: ( i ) a reduced promoter leakiness [ 2 , 4 , 51 ], ( ii ) unsaturated levels of AAA+ proteases ClpXP [ 9 ], and ( iii ) small values of Hill coefficients [ 7 – 9 , 16 ]. Because the impact of the variation in the values of the Hill coefficients and leakiness parameters on the bistability region in the A model is carefully analyzed in [ 16 ], we validate variations in these parameters for the S model only.…”

Section: Modelsmentioning

confidence: 99%

“…Another important parameter ( γ ) is the promoter leakiness [ 2 , 4 , 51 ]. To this end, very small dimensionless values of the corresponding leakiness parameters are used in the relevant modeling studies [ 16 , 17 ].…”

Section: Modelsmentioning

confidence: 99%

“…The tightness of the promoter control has been reported in the literature, e.g. , see [ 2 , 4 , 12 , 51 ], and the reduction in the promoter leakiness is the subject of active ongoing research with the promise to reduce this leakiness even further dramatically [ 51 ].…”

Section: Modelsmentioning

confidence: 99%

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Quorum-Sensing Synchronization of Synthetic Toggle Switches: A Design Based on Monotone Dynamical Systems Theory

Nikolaev

Sontag

2016

PLoS Comput Biol

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Synthetic constructs in biotechnology, biocomputing, and modern gene therapy interventions are often based on plasmids or transfected circuits which implement some form of “on-off” switch. For example, the expression of a protein used for therapeutic purposes might be triggered by the recognition of a specific combination of inducers (e.g., antigens), and memory of this event should be maintained across a cell population until a specific stimulus commands a coordinated shut-off. The robustness of such a design is hampered by molecular (“intrinsic”) or environmental (“extrinsic”) noise, which may lead to spontaneous changes of state in a subset of the population and is reflected in the bimodality of protein expression, as measured for example using flow cytometry. In this context, a “majority-vote” correction circuit, which brings deviant cells back into the required state, is highly desirable, and quorum-sensing has been suggested as a way for cells to broadcast their states to the population as a whole so as to facilitate consensus. In this paper, we propose what we believe is the first such a design that has mathematically guaranteed properties of stability and auto-correction under certain conditions. Our approach is guided by concepts and theory from the field of “monotone” dynamical systems developed by M. Hirsch, H. Smith, and others. We benchmark our design by comparing it to an existing design which has been the subject of experimental and theoretical studies, illustrating its superiority in stability and self-correction of synchronization errors. Our stability analysis, based on dynamical systems theory, guarantees global convergence to steady states, ruling out unpredictable (“chaotic”) behaviors and even sustained oscillations in the limit of convergence. These results are valid no matter what are the values of parameters, and are based only on the wiring diagram. The theory is complemented by extensive computational bifurcation analysis, performed for a biochemically-detailed and biologically-relevant model that we developed. Another novel feature of our approach is that our theorems on exponential stability of steady states for homogeneous or mixed populations are valid independently of the number N of cells in the population, which is usually very large (N ≫ 1) and unknown. We prove that the exponential stability depends on relative proportions of each type of state only. While monotone systems theory has been used previously for systems biology analysis, the current work illustrates its power for synthetic biology design, and thus has wider significance well beyond the application to the important problem of coordination of toggle switches.

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Section: Resultsmentioning

confidence: 99%

Section: Modelsmentioning

confidence: 99%

Section: Modelsmentioning

confidence: 99%

Section: Modelsmentioning

confidence: 99%

See 2 more Smart Citations

Quorum-Sensing Synchronization of Synthetic Toggle Switches: A Design Based on Monotone Dynamical Systems Theory

Nikolaev

Sontag

2016

PLoS Comput Biol

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“…A combinatorial TetR repressed promoter library has been described previously with varying operator multiplicity and location resulting in promoters with leaky expression in the repressed state spanning more than two orders of magnitude (Murphy et al , 2007). The strength of repressor binding to its operator sites can affect promoter leakiness and can be tuned through mutation of the operator DNA sequence or the DNA-binding domain of the repressor protein (Nichols & Matthews, 1997; Penumetcha et al , 2010). Another way with which basal transcription can be tuned is through the co-expression of the target gene from a constitutive promoter as well as a regulated promoter, although this will also result in an increase in gene expression level in the induced state.…”

Section: Introductionmentioning

confidence: 99%

Tuning the dials of Synthetic Biology

et al. 2013

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Synthetic Biology is the ‘Engineering of Biology’ – it aims to use a forward-engineering design cycle based on specifications, modelling, analysis, experimental implementation, testing and validation to modify natural or design new, synthetic biology systems so that they behave in a predictable fashion. Motivated by the need for truly plug-and-play synthetic biological components, we present a comprehensive review of ways in which the various parts of a biological system can be modified systematically. In particular, we review the list of ‘dials’ that are available to the designer and discuss how they can be modelled, tuned and implemented. The dials are categorized according to whether they operate at the global, transcriptional, translational or post-translational level and the resolution that they operate at. We end this review with a discussion on the relative advantages and disadvantages of some dials over others.

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Bioprocess development to produce a hyperthermostable S‐methyl‐5′‐thioadenosine phosphorylase in Escherichia coli

Schollmeyer,

Waldburger,

Njo

et al. 2023

Biotech & Bioengineering

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Nucleoside phosphorylases are important biocatalysts for the chemo‐enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S‐methyl‐5′‐thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide‐spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24‐well plates using a fed‐batch medium. Subsequently, HCD cultivations were performed using E. coli BL21‐Gold cells, by employing a glucose‐limited fed‐batch strategy. Comparing different growth rates in stirred‐tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500‐mL scale, final cell dry weights of 87.1–90.1 g L−1 were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg−1 (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.

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Improving the Lac System for Synthetic Biology

Cited by 22 publications

References 13 publications

Quorum-Sensing Synchronization of Synthetic Toggle Switches: A Design Based on Monotone Dynamical Systems Theory

Quorum-Sensing Synchronization of Synthetic Toggle Switches: A Design Based on Monotone Dynamical Systems Theory

Tuning the dials of Synthetic Biology

Bioprocess development to produce a hyperthermostable S‐methyl‐5′‐thioadenosine phosphorylase in Escherichia coli

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