Design and Construction of a Switchable Bistable

Switch in Yeast

Yaoyu Yang, Eric Klavins Department of Electrical Engineering University of Washington, Seattle, WA

Abstractâ??The genetic bistable switch is a fundamental build- 1"

ing block for engineering synthetic living systems. In synthetic biology, researchers have designed and constructed rudimentary bistable switches in E. coli and in mammalian cells. However, it remains challenging to construct a reusable, modular, switchable bistable switch in eukaryotes. Here we describe a design for such

a bistable switch in S. cerevisiae (yeast), develop a predictive 2"

mathematical model, and present experimental results demon-

FBox'

GEV" 3" GAVNY"

strating the functionality, and in particular, the switchability, of the switch.

I. INTRODUCTION

G! E! V!

G. GAL4 DNA binding domain!

E. Estrogen Receptor!

V. VP16!

Y!

G! A! V! N!

G. GAL4 DNA binding domain!

A. Auxin degron IAA17.T2!

V. VP16!

N. Nuclear localization signal (2x SV40)!

Y. EYFP!

The genetic bistable switch is arguably a key building block for engineering synthetic living systems as it forms an addressable cellular memory unit and could be used for controlling cell states [1]. Researchers have designed and constructed bistable switches resulting in various designs and implementations from E. coli to mammalian cells over the past 15 years of synthetic biology [1], [3], [4]. However, currently there has not been a robust inducible bistable switch in yeast. Here, we introduce a design and implementation of a switchable bistable switch in yeast. Specifically, the switch can can be switched to a high state by adding β-estradiol and switched to a low state by exposure to the plant hormone auxin. Experimental results also show that the switch is able to maintain either its low or high state without the continued presence of these small molecules.
II. A BISTABLE SWITCH DESIGN IN YEAST
The design incorporates a positive feedback loop to achieve bistability and uses two other components to make the cir- cuit switchable via exposure to small molecules. The circuit diagram is shown in Fig. 1. The circuit consists of three components: GEV, a β-estradiol sensitive transcriptional acti-

Fig. 1. The bistable switch design in yeast 1) Circuit diagram of the yeast bistable switch. 2) Construction scheme of GEV where it consists of three protein subdomains. GAL4 DNA binding domain, which recognizes pGAL1 promoter; the estrogen receptor domain interacts with β-estradiol, which makes the activator inducible by β-estradiol; VP16 is an transcriptional activation domain. 3) Construction scheme of GAVNY where it consists of four protein subdomains and a fluorescence tag. IAA17.T2 represents for T2 domain of Aux/IAA17 protein from Arabidopsis thaliana [2] and nuclear localization signal is responsible for brining the protein into nucleus to perform transcriptional control function.

each constitutively expressed through promoters pACT1 and pGPD. The GAVNY is fluorescent, as it is fused to an EYFP reporter. The functionality of this design is first explored by the following mathematical models.
III. MATHEMATICAL MODELS
This circuit can be modeled through a biochemical reaction network derived from the circuit design. Assuming mass action kinetics, we get the following reaction rate equation for GAVNY based on the assumption that other related species are in quasi-steady-state:
vator developed and widely used in yeast [5], which activates promoter pGAL1 in the presence of β-estradiol; GAVNY, a novel auxin sensitive transcriptional activator that we built and

dA λa An + λb v

=

dt 1 + ρa An + ρb v

αf γaf Au

â?? λf + γaf Au

â?? δa A (1)

activates pGAL1 forming the positive feedback loop; FBox from Arabidopsis thaliana [2], which degrades GAVNY when induced with auxin. We hypothesize that the circuit behaves as follows. When induced with β-estradiol, GAVNY is produced by the activation of GEV and stays at a high state owing to the positive feedback loop even when the β-estradiol is removed; when induced with auxin, GAVNY is completely degraded by the FBox and stays at low state even when auxin is removed. The GEV and FBox in this circuit are

Here A represents the concentration of GAVNY, v represents the concentration of β-estradiol and u represents the concentra- tion of auxin. λa and ρa are parameters modeling the activation strength of GAVNY by itself, n is the Hill coefficient, λb and ρb are parameters responsible for the activation strength of GAVNY by GEV that induced by β-estradiol, αf , γaf and λf are parameters that capture the degradation of GAVNY induced by auxin, δa models the basal degradation and dilution rate of GAVNY.

1"

200"

u"="0,"v"="1" u"="1,"v"="0" u"="0,"v"="0"

1" 5000"

100"nM" β:estradiol"

20"μM"" auxin"

no"inducer"

0"

2" 200"

0" 300" 600" 900"

1000"

2"

0" 5" 17" 37"

u"="0,"v"="1" u"="0,"v"="0"

0"

0" 300" 600" 900"

t"

5000" :

:

1000":

100"nM"

β:estradiol" no"inducer"

0" 5" 17" 37"

Time"(hr)"

Fig. 2. Numerical simulations demonstrating bistability and switchability..

1) The initial condition is A = 0, the system is first induced with β-estradiol shown as u = 0, v = 1 and the system is switched to the high state. Then at t = 300, β-estradiol is removed and auxin is added, simulated as u =

1, v = 0, the system is switched back to the low state. After t = 600, auxin is removed and there is no inducer around, simulated as u = 0, v = 0, and the system is still kept at the low state. 2) The system is first induced with β- estradiol and switched to the high state. Then β-estradiol is removed and the system is still kept at the high state. Parameters used in both simulations are n = 2, λa = 0.08, ρa = 0.01, δa = 0.045, αf = 8, γaf = 0.113, λf =

1, λb = 1, Ï?b = 1.

Analysis show that this system can exibit bistability under certain parameter constraints. In Fig. 2, we show simulation results that demonstrate the bistability and switchability. of this system.
IV. EXPERIMENTAL CONSTRUCTION AND RESULTS
The bistable switch was constructed following the design shown in Fig. 1. pACT1-GEV, pGAL1-GAVNY, and pGPD- FBox were first constructed on ampicillin resistant plasmids, then digested and integrated into the yeast genome sequen- tially.
The preliminary results for validating the functionality of this inducible bistable switch are shown in Fig. 3. The yeast cells were cultured in 30 C and fluorescence measurements were taken using a flow cytometer. The preliminary experi- mental results show that the circuit was induced to a high state with the addition of 100 nM β-estradiol and switched to a low state by adding 20 µM auxin. The data also shows that the circuit was able to keep at the low state when auxin was diluted out and remained in the high state when β-estradiol was diluted out. These results demonstrate the bistability and switchability of this circuit.
V. CONCLUSION
In this abstract, we presented an inducible bistable switch design and implementation in yeast. A design scheme for the inducible bistable switch was described and a mathematical model was developed to explore the functionality of this

Fig. 3. Experimental results demonstrating bistability and switchability.

1) The circuit was first induced with 100 nM β-estradiol at t = 0, the fluorescence measurement at t = 5 shows that the circuit switched to the high state. Also at t = 5, β-estradiol was diluted out and 20 µM auxin was added, after 12 hrs, data shows that the circuit switched to the low state. Auxin was diluted out at t = 17 and data after 20 hrs shows that the circuit kept at low state. 2) The circuit was first induced with 100 nM β-estradiol at t = 0, data at t = 5 shows that the circuit switched to high state. At t = 5, β-estradiol was diluted out and later fluorescence measurements at t = 17, t = 37 show that the circuit kept at high state.

design. An experimental implementation was constructed and preliminary results were presented. The results showed a bistable switch in yeast that can be switched into high and low state using two different inducers β-estradiol and auxin. This switch is able to remain at either states for the time duration tested in our preliminary experiments. These results thus describe the first switchable synthetic bistable switch in S. cerevisiae.
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