designing synthetic gene networks using artificial transcription factors in yeast
TRANSCRIPT
Designing Synthetic Gene Networks Using Artificial Transcription Factors in Yeast!Matthew Baron1, Matthew Farnitano1, Cameron Kim1, Hyun Soo Kim1, Ashley Reid1, Janan Zhu1, Charles Cooper2, !
Dr. Nicolas Buchler3,4,5, Dr. Charles Gersbach3,5,6!1iGEM Team Member, 2iGEM Team Mentor, 3Faculty Advisor, 4Department of Biology, Duke University, Durham, NC, 5Institute for Genome
Sciences & Policy, Duke University, Durham, NC, 6Department of Biomedical Engineering, Duke University, Durham, NC!!
Synthetic gene circuits have the potential to revolutionize gene therapies and bio-industrial methods by allowing predictable, customized control of gene expression. Bistable switches and oscillators, key building blocks of more complex gene networks, have been constructed using naturally occurring and well-characterized regulatory elements. In order to expand the versatility and variety of these circuits, our long term goal is to design and construct gene networks using artificial transcription factors (ATFs). The ATFs are of two classes: inhibitory TALE proteins and a catalytically inactive dCas9 protein with small guide RNA elements, each orthogonal to the yeast genome. Using mathematical modeling, we determined the parameters expected to create bistability, using tandem binding site kinetics to achieve cooperativity. Based on these results, we assembled a library of plasmids containing ATFs, binding sites, regulatory elements, and fluorescent reporters. We then integrated these genes into the genome of Saccharomyces cerevisiae and are currently characterizing them using flow cytometry.!
Abstract!
Introduction & Background!
Experimental Design and Procedures! Results!
Conclusions and Future Directions!
Acknowledgements!
The bacterial “toggle switch” was developed in 2000 with the ability to flip between two distinct genetic states with the addition of an external inducer (Gardner et al. 2000).The synthetic gene construct consisted of two repressor proteins linked in mutual repression to generate a positive feedback loop. These repressors act as dimers and achieve a bistable response through cooperative binding. That same year, the “repressilator” synthetic gene circuit was created, consisting of three linked repressors to generate oscillations in gene expression (Elowitz & Liebler, 2000). !
Repressor 1! Repressor 2!
Repressor 1!
Repressor 2!
Repressor 3!
Figure 1: Schematics of the genetic toggle switch (left) and repressilator circuit (right). Circles indicate protein product and lines with solid bars at end indicate repression. !
In order to achieve a bistable response, these foundational circuits utilized existing bacterial repressor proteins that exhibit inherent cooperativity through protein dimerization. Synthetic gene circuits have built upon using these same proteins and similar modules to engineer higher order circuits, such as feed-forward loops and robust oscillators, but have since stalled due to the limited number of characterized parts with customizable features, particularly the binding sequence. In order to generate synthetic gene circuits using endogenous promoters and sequences, we utilize artificial transcription factors that can target a user-defined sequence. Most of these proteins act as monomers, and will not experience cooperativity in their current form. In order to achieve cooperativity, we are using tandem binding sites in the promoter region. The multiple binding of repressors to the promoter will allow for a cooperative response that can be utilized in designing a bistable toggle switch. Bistable toggle switches are important regulatory motifs in cell cycle, differentiation, and other cell-fate decisions, and can also be utilized in potential gene therapies to precisely administer a therapeutic protein through an inducer. We are focusing our gene circuits in Saccharomyces cerevisiae as such a circuit has yet to be built in yeast. !!The artificial transcription factors (ATFs) that we use are Transcription Activator-like Effectors (TALEs) and the CRISPR/Cas9 system (Clustered Regularly Interspaced Palindromic Repeats). These DNA binding proteins can be customized to target almost any DNA sequence. TALEs are derived from a plant pathogen Xanthomonas sp. and the CRISPR/Cas9 system is adapted from a bacterial adaptive immunity pathway. TALEs use protein motifs to bind the DNA, while the CRISPR/Cas9 system uses a small guide RNA molecule that binds the DNA while the Cas9 protein associates with the RNA molecule. Instead of utilizing Cas9 which is a nuclease, we use a catalytically dead Cas9 (dCas9) that acts as a roadblock to transcriptional activation. By utilizing these proteins, we can design orthogonal gene circuits that can be translated across multiple domains of life. !
Figure 2: TALE protein binding DNA (left) and the Cas9 protein binding a guide RNA and target DNA sequence (right, DiCarlo et al., 2013)!
Constructing a Library of Gene Circuit Parts!• We created a parts library with two kinds of constructs, reporters and
repressors.!• Reporters contain repressor binding sequences in between TEF1pr
and the yEVenus gene (Murphy et al., 2007).!• We’ve constructed 12 out of 18 plasmids (BBa_K1204013 –
BBa_K120424) that vary in binding sequence number, length, and content.!
• Repressor plasmids provide inducible expression of TALE and iCas9-gRNA complexes tailored for a specific binding sequence.!
• TALE proteins have mCherry tags to indicate expression.!• The large variety in binding sequence constructs allow us to observe what
facilitates the greatest cooperativity in binding.!• The library facilitates combining different repressor and reporter
components for the construction of complex gene circuits.!!
Mathematical Modeling!!!
!!
I. Thermodynamic Model of Cooperative Repression !• Our thermodynamic model was largely based on the models shown in "Transcriptional regulation by the numbers: models" (Bintu et al., 2005). ! !• We assumed gene expression level is proportional to the probability of RNA Polymerase (RNAP) binding to the DNA of interest (pbound) (Eq 1).!• Using Boltzmann weights of all possible states, pbound in the absence and presence of any repressors was derived. (Eq. 2 and 3)!• Freg terms (Eq.3) accounted for how strongly the repressor lowers pbound!
• The effects of number of repressor binding sites and strength of repressor binding (Δεrd) on cooperativity were demonstrated. (Eq. 4)!• Apparent Hill coefficients were obtained from our non-Hill Function model.!!• Results showed that increasing number of binding sites and balanced repressor binding strengths increased the cooperativity.!
• Kinetic model was used to study conditions necessary for a system to be bistable. !
• Our key assumptions were: !1. No active degradation of repressors (only diluted due to cell growth)!2. Five repressor binding sites!3. Basal level gene expression at maximum repression (“leakiness”)!• A plot of nullclines and trajectories confirmed that our model could
demonstrate a bistable system when given appropriate parameters.!• Effect of promoter strengths, repressor binding strengths and basal
rate of gene expression (leakiness of promoters) on bistable region was demonstrated.!
!!
II. Kinetic Model of Bistable Toggle Switch
Design of Synthetic Gene Circuit to test Cooperative Response!• The fluorescent protein yEVenus is used to report the cooperative
response. Between the promoter and yEVenus are either 1, 3, or 5 identical binding sites of length 16 or 20 bp that can be bound by the TALE-mCherry fusion(no activating or repressing domains) or guide RNA for the CRISPR/dCas9 system.!
• The Z4EV zinc finger transcription factor is initially in the cytoplasm being sequestered by HSP90. When Z4EV is induced by the addition of β-estradiol, the HSP90 dissociates and it translocates to the nucleus (McIsaacs et al., 2012)!
• Once in the nucleus, Z4EV activates the transcription of the TALE or dCas9 protein, which binds the reporter construct and blocks the transcription by roadblocking RNA polymerase. !
• Fluorescence levels can be analyzed using flow cytometry. !• If cooperativity is observed, mathematical modeling of estimated
parameters can guide the selection of TALE or CRISPR/Cas9 cassettes to design a toggle switch. !
TALE production is induced under the expression of β-estradiol!
!• When yeast are grown in media containing β-
estradiol, there is a 4-fold induction in TALE-mCherry production as measured by flow cytometry after 6 hours for mCherry.!
• Induction curves and time course demonstrate the presence of increased levels of mCherry production with β-estradiol. !
!Figure 7: Change in TALE-mCherry with β-estradiol induction!
Figure 8: Time course for Z4EV promoter induction with β-estradiol (left) and dose response for Z4EV promoter with concentrations of β-estradiol from 0.1 nM to 1000 nM!
Under the expression of a constitutive promoter, yEVenus production increases!• Under the expression of the ACT1 promoter, the insertion of binding sites reduced
yEVenus expression to near background levels. With the TEF1pr, the insertion of binding sites produces YFP expression levels to that of the ACT1pr without binding sites. !
Figure 9: Expression of yEVenus with 3x binding site from ACT1 promoter (left) versus TEF1 promoter (right)!
• We have constructed a library of parts, including TALEs, guide RNAs, dCas9, and fluorescent reporters. !
• TALE-mCherry and dCas9 production is inducible under β-estradiol in our yeast strain. !
• Using the TEF1 promoter, we can achieve a dynamic range of fluorescence values. !
• We plan to test the ATF system to ascertain cooperativity of multiple binding sites and generate parameters essential to our circuit design and modeling.!
• Guided by mathematical modeling, we plan to select parts to make our yeast toggle switch and other gene circuits. !
Effect of Changing Number of Binding Sites and Binding Strengths on the Apparent Hill Coefficient
n=4.8635 n=2.9929 n=1.0 n=4.87
n=4.86
n=4.29
n=4.40 n=4.52
n=4.72
Log(V)
Nullclines and Trajectories of Bistable SystemLog(U)
Bistable Regions for Unbalanced Binding Strengths Bistable Regions for Leaky Promoter
Log(Kd, repressor1)
Log(
K d, r
epre
ssor
2)
Log(
K d, r
epre
ssor
2)
Effect of Changing Binding Strength and Basal Expression Level on the Bistable RegionLog(Kd, repressor1)
We thank the Lord-Alstadt Foundation for funding the Duke iGEM team and Duke’s Undergraduate Research Support Office for providing travel grants to us.!
References!1. Gardner, T., Cantor, C., Collins, J. Nature, 403, 339-342. (2000)!2. Elowitz, M., & Liebler, S., Nature, 403, 335-338. (2000)!3. DiCarlo, J., et al., Nucleic Acids Research, 41, pp. 4336-4343. (2013)!4. McIsaacs, R., et al., Nucleic Acids Research, 41, e. 57. (2013)!5. Bintu, L., et al., Curr Opin Genet Dev, 15, 125-135. (2005)!6. Murphy, K.F., et al., PNAS, 104, 12726-12731. (2007)!