Name: rapid synthesis and screening of chemically activated transcription factors with gfp-based reporters
Uploaded: 2013-11-26T20:00:00
Duration: 9 min 22 s
Description: Watch this Scientific Journal Video about Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters at JoVE.com

R.S.M. acknowledges funding from the NSF Graduate Research Fellowship. M.B.N. acknowledges funding from a Lewis-Sigler Fellowship and the endowed gift of Peter Lewis. D.B. acknowledges funding from the NIH (GM046406) the National Institute of General Medical Sciences Center for Quantitative Biology (GM071508). We acknowledge Christina DeCoste for assistance with flow cytometry experiments.

Figure 2 provides a schematic of protocol sections 1-3.
1. Creation of ATFs
<ol>
	<li>Create primers for PCR amplifying DNA binding domain of interest. Homology to the ACT1 promoter and hormone receptor (as described in Figures 3 and 4) is added through PCR to facilitate correct recombination into the strain yMN15.</li>
	<li>PCR amplify DBD of interest using a high fidelity polymerase.</li>
	<li>Transform &#62;1 &#181;g of purified PCR product into yeast using the lithium acetate method13.</li>
	<li>Following transformation, spin cells at top speed for 15 sec&#160;in microcentrifuge and remove transformation mix.</li>
	<li>Resuspend in ~200 &#181;l&#160;of sterile water and plate on YPD.</li>
	<li>The next day, replica plate from YPD onto 5-FOA plates (5-FOA plates are made as described in the Cold Spring Harbor Yeast Manual)13.</li>
	<li>Allow growth for 2-4 days, and restreak at least 5-10 colonies onto YPD. Perform a colony PCR using the primers in Table 1 and sequence to verify inclusion.</li>
</ol>
2. Creation of ATF Plasmid Reporter
<ol>
	<li>Determine the binding site of the ATF (most likely to come from the literature).</li>
	<li>Order desired binding region as oligos (or Ultramers, if needed). Order as top and bottom strands with correct overhangs for NotI &#38; XbaI as shown in Table 2.</li>
	<li>Dilute oligos to equimolar concentrations (100 &#181;M).</li>
	<li>Phosphorylate using T4 Polynucleotide Kinase and then anneal in a thermocycler. The reaction conditions are in Table 3.</li>
	<li>Digest ~1-2 &#181;g of pMN8 with NotI-HF &#38; XbaI for 1 hr at 37 &#176;C (reaction conditions in Table 4).</li>
	<li>Gel purify the backbone band.</li>
	<li>Dilute the purified backbone to 10 ng/&#181;l.</li>
	<li>Dilute the double-stranded, phosphorylated binding site to 5x the molar concentration of backbone per &#181;l.</li>
	<li>Using T4 DNA Ligase, ligate the binding sites into the digested backbone at room temperature for 30 min&#160;(Table 5).</li>
	<li>For transformation, add half the ligation reaction to 50 &#181;l&#160;of standard, chemically competent Escherichia coli&#160;such as XL1-Blue&#160;or DH5-Alpha.</li>
	<li>Heat shock cells for 45 sec&#160;in a 42 &#176;C water bath and then place on ice for 2 min.</li>
	<li>Add 900 &#181;l&#160;of SOC medium to transformation reaction.</li>
	<li>Grow at 37 &#176;C for 1 hr on a roller drum.</li>
	<li>Spread 100-200 &#181;l cells per LB+AMP (100 &#181;g/ml) plate and incubate overnight.</li>
	<li>Grow up&#160;E. coli in LB+AMP liquid and harvest plasmid DNA. Sequence verify new reporter plasmid from ligation colonies using the&#160;primer 5&#39;-GTACCTTATATTGAATTTTC-3&#39;.</li>
	<li>Transform new reporter plasmid into ATF-containing yeast strain from Section 1 using lithium acetate transformation.</li>
</ol>
3. Quantify the Effect of ATF Induction on Gene Expression Using GFP Reporter
Preparing the samples:
<ol>
	<li>Make 25 mM &#946;-estradiol stock (10 ml&#160;of ethanol + 0.0681 g&#160;of &#946;-estradiol). Keep refrigerated.</li>
	<li>Grow ATF + Reporter-containing strain overnight in 5ml&#160;SC-URA.</li>
	<li>Obtain nine 250 ml&#160;shake flasks and add 25 ml&#160;SC-URA to each.</li>
	<li>Add 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 &#181;M, or 10 &#181;M &#946;-estradiol. This is most easily done by making 10-fold serial dilutions of the 25 mM &#946;-estradiol stock. For a final concentration of 10 &#181;M &#946;-estradiol, add 10 &#181;l&#160;of the 25 mM &#946;-estradiol stock to 25 ml&#160;of SC-URA.</li>
	<li>Add 125 &#181;l&#160;of overnight cultures to flasks (1:200 dilution).</li>
	<li>For the 2 remaining flasks, inoculate with a constitutive GFP producing strain and a strain lacking GFP. These are needed for calibrating the flow cytometer.</li>
	<li>Shake at 200 rpm at 30 &#176;C for 12-18 hr. 
	Note: The optimal incubation time is determined by identifying when GFP induction no longer changes following addition of &#946;-estradiol.</li>
	<li>Take 500 &#181;l&#160;of each culture and spin down in separate 1.7 ml microcentrifuge tubes.</li>
	<li>Aspirate SC-URA.</li>
	<li>Wash once in 1 ml&#160;PBS+0.1% Tween-20 and aspirate.</li>
	<li>Add 1 ml&#160;of PBS+0.1% Tween-20 and resuspend.</li>
	<li>Add 1 ml&#160;of PBS+0.1% Tween-20 to Falcon tubes.</li>
	<li>Add the resuspended cells to 5 ml&#160;tubes (final volume = 2 ml) (can be stored at 4 &#176;C for several days).</li>
	<li>Optional: sonicate cells lightly to breakup any clumped cells. 
	Flow cytometry analysis: Overview: The fluorescence of 10,000-100,000 cells is typically analyzed per sample. Data can be processed either in FlowJo or using custom scripts in MATLAB or R. Make sure to calibrate voltage of the GFP channel using GFP positive and negative controls. Once the FCS files are exported, a script such as the one in Figure 6 along with the function fca_readfcs (<a href="http://www.mathworks.com/matlabcentral/fileexchange/9608-fcs-data-reader">http://www.mathworks.com/matlabcentral/fileexchange/9608-fcs-data-reader</a>) can be used to process the data.</li>
	<li>Perform flow cytometry.
	<ol>
		<li>Use forward scatter (FSC) and side scatter (SSC) to identify yeast cells.</li>
		<li>Set the flow rate to ~3,000 cells/sec.</li>
		<li>Measure GFP (FITC channel).</li>
		<li>Once data are collected, export .FCS files</li>
	</ol>
	</li>
	<li>Load MATLAB.</li>
	<li>Move the .FCS files, fcs_readfcs.m, and a script similar to that from Figure 6 to the same folder.</li>
	<li>Change the present working directory to the folder containing the data and scripts.</li>
	<li>Run the script from Figure 6 (note: the legend has been manually entered to produce what&#39;s seen in Figure 7).</li>
</ol>
4. Quantify the Effect of ATF Induction on Cell Growth
<ol>
	<li>From frozen stocks, streak out both (1) an ATF-containing strain and (2) an ATF-lacking strain (such as yMN15) onto separate YPD plates.</li>
	<li>After 2 days of growth, inoculate separate culture tubes containing 5 ml&#160;of YPD liquid with each strain and grow overnight at 30 &#176;C.</li>
	<li>Perform 10-fold serial dilutions of a 0.25 mM &#946;-estradiol stock solution up to 10,000-fold (0.025 &#181;M &#946;-estradiol) in 100% ethanol.</li>
	<li>Add 40 &#181;l&#160;of overnight cultures to 10 ml&#160;of YPD liquid (1-250 dilution).</li>
	<li>For each strain, add 96 &#181;l&#160;of diluted cells to 18 wells of a 96-well plate.</li>
	<li>Add 4 &#181;l&#160;of &#946;-estradiol to the cells. An essential point is to make sure each well has the same amount of total ethanol. (Alternatively, a more concentrated stock of &#946;-estradiol can be used and subsequently diluted to the point the effect of ethanol on growth is not measurable).</li>
	<li>Perform in triplicate. Three of the wells for each strain should be ethanol-only controls.</li>
	<li>Cover the 96-well plate in gas permeable membrane.</li>
	<li>Monitor growth (OD600) in plate reader.</li>
	<li>Quantify the growth rates using any number of standard methods such as finding the maximum slope. 
	Note: We&#39;ve had good success with the open source software package grofit14, which runs in the R programming environment. It is worth mentioning that while growth rates computed from 96-well plate assays compared to shake-flasks (25 ml&#160;cultures) are not necessarily the same (due to both differences in physiology and in instrumentation), we&#39;ve observed that the relative growth rates are similar (though this may not always be the case).</li>
</ol>
5. Create Inducible Genomic Alleles
<ol>
	<li>Obtain pMN10, a noncentromeric plasmid with the ATF-responsive promoter from pMN8 fused to KanMX (in the opposite orientation).</li>
	<li>Restriction digest the vector backbone and gel extract as in Section 2.</li>
	<li>Anneal, phosphorylate, and ligate oligos containing appropriate binding sites as described in Section 2.</li>
	<li>Sequence verify. This plasmid will now serve as a DNA template for creating the inducible allele.</li>
	<li>Obtain the yeast strain expressing the corresponding ATF of interest.</li>
	<li>PCR amplify the KanMX-Promoter cassette (~2 kb) using primers from Table 6.</li>
	<li>Transform the PCR product into the ATF-expressing strain using the lithium acetate method.</li>
	<li>Plate on YPD and replica plate onto YPD+ G418 the following day.</li>
	<li>Confirm appropriate insertion of the promoter using the forward primer (5&#39;-CTGCAGCGAGGAGCCGTAAT-3&#39;) and a reverse primer internal to the gene whose promoter has been replaced. The reverse primer is typically designed to be between 200-300 bp downstream of the first ATG15. The final PCR product is ~1.2 kb.</li>
</ol>

<ol>
	<li>McIsaac, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+gene+expression+perturbation+systems+with+rapid,+tunable,+single-gene+specificity+in+yeast.">Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast.</a> Nucleic acids res. 41, e57 (2013).</li><li>McIsaac, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast-acting+and+nearly+gratuitous+induction+of+gene+expression+and+protein+depletion+in+Saccharomyces+cerevisiae.">Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae.</a> Mol. Biol. cell. 22, 4447-4459 (2011).</li><li>Louvion, J. F., Havaux-Copf, B., Picard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+GAL4-VP16+to+a+steroid-binding+domain+provides+a+tool+for+gratuitous+induction+of+galactose-responsive+genes+in+yeast.">Fusion of GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast.</a> Gene. 131, 129-134 (1993).</li><li>Littlewood, T. D., Hancock, D. C., Danielian, P. S., Parker, M. G., Evan, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+oestrogen+receptor+ligand-binding+domain+as+an+improved+switch+for+the+regulation+of+heterologous+proteins.">A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins.</a> Nucleic acids res. 23, 1686-1690 (1995).</li><li>McIsaac, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+Analysis+of+mRNA+Molecules+Using+Fluorescence+In+Situ+Hybridization+in+Saccharomyces+cerevisiae.">Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae.</a> J. Vis. Exp. (76), e50382 (2013).</li><li>McIsaac, R. S., Petti, A. A., Bussemaker, H. J., Botstein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation-based+analysis+and+modeling+of+combinatorial+regulation+in+the+yeast+sulfur+assimilation+pathway.">Perturbation-based analysis and modeling of combinatorial regulation in the yeast sulfur assimilation pathway.</a> Mol. Biol. cell. 23, 2993-3007 (2012).</li><li>Belli, G., Gari, E., Piedrafita, L., Aldea, M., Herrero, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+activator/repressor+dual+system+allows+tight+tetracycline-regulated+gene+expression+in+budding+yeast.">An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast.</a> Nucleic acids res. 26, 942-947 (1998).</li><li>Baron, U., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+repressor-based+system+for+regulated+gene+expression+in+eukaryotic+cells:+principles+and+advances.">Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances.</a> Meth. Enzymol. 327, 401-421 (2000).</li><li>Kodama, S., Okada, K., Akimoto, K., Inui, H., Ohkawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+aryl+hydrocarbon+receptors+for+bioassay+of+aryl+hydrocarbon+receptor+ligands+in+transgenic+tobacco+plants.">Recombinant aryl hydrocarbon receptors for bioassay of aryl hydrocarbon receptor ligands in transgenic tobacco plants.</a> Plant Biotechnol. J. 7, 119-128 (2009).</li><li>Hickman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+regulation+of+sulfur+and+phospholipid+metabolism+reflects+the+importance+of+methylation+in+the+growth+of+yeast.">Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast.</a> Mol. Biol. cell. 22, 4192-4204 (2011).</li><li>Taxis, C., Stier, G., Spadaccini, R., Knop, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+protein+depletion+by+genetically+controlled+deprotection+of+a+dormant+N-degron.">Efficient protein depletion by genetically controlled deprotection of a dormant N-degron.</a> Mol. Syst. Biol. 5, 267 (2009).</li><li>Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., Kanemaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+auxin-based+degron+system+for+the+rapid+depletion+of+proteins+in+nonplant+cells.">An auxin-based degron system for the rapid depletion of proteins in nonplant cells.</a> Nat. methods. 6, 917-922 (2009).</li><li>Burke, D. J., Amberg, D. C., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. , (2005).</li><li>Kahm, M., Hasenbrink, G., Lichtenberg-Frate, H., Ludwig, J., Kschischo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=grofit:+Fitting+Biological+Growth+Curves+with+R.">grofit: Fitting Biological Growth Curves with R.</a> J. Stat. Softw. 33, 1-21 (2010).</li><li>Rozen, S., Skaletsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer3+on+the+WWW+for+general+users+and+for+biologist+programmers.">Primer3 on the WWW for general users and for biologist programmers.</a> Methods Mol. Biol. 132, 365-386 (2000).</li><li>Khalil, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+biology+framework+for+programming+eukaryotic+transcription+functions.">A synthetic biology framework for programming eukaryotic transcription functions.</a> Cell. 150, 647-658 (2012).</li><li>Chockalingam, K., Chen, Z., Katzenellenbogen, J. A., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+specific+receptor-ligand+pairs+for+use+in+the+creation+of+gene+switches.">Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 5691-5696 (2005).</li><li>McLachlan, M. J., Chockalingam, K., Lai, K. C., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+orthogonal+ligand+specificity+in+a+single+scaffold.">Directed evolution of orthogonal ligand specificity in a single scaffold.</a> Angewandte Chemie. 48, 7783-7786 (2009).</li><li>Jorgensen, P., Nishikawa, J. L., Breitkreutz, B. J., Tyers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+identification+of+pathways+that+couple+cell+growth+and+division+in+yeast.">Systematic identification of pathways that couple cell growth and division in yeast.</a> Science. 297, 395-400 (2002).</li><li>Sopko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+pathways+and+phenotypes+by+systematic+gene+overexpression.">Mapping pathways and phenotypes by systematic gene overexpression.</a> Mol. cell. 21, 319-330 (2006).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nat. biotechnol. 20, 1041-1044 (2002).</li><li>Polstein, L. R., Gersbach, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-inducible+spatiotemporal+control+of+gene+activation+by+customizable+zinc+finger+transcription+factors.">Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors.</a> J. Am. Chem. Soc. 134, 16480-16483 (2012).</li><li>Losi, A., Gartner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Old+chromophores,+new+photoactivation+paradigms,+trendy+applications:+flavins+in+blue+light-sensing+photoreceptors.">Old chromophores, new photoactivation paradigms, trendy applications: flavins in blue light-sensing photoreceptors.</a> Photochem. Photobiol. 87, 491-510 (2011).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat. Neurosci. 8, 1263-1268 (2005).</li><li>Gradinaru, V., Mogri, M., Thompson, K. R., Henderson, J. M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+deconstruction+of+parkinsonian+neural+circuitry.">Optical deconstruction of parkinsonian neural circuitry.</a> Science. 324, 354-359 (2009).</li><li>Milias-Argeitis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+feedback+for+in+vivo+regulation+of+a+gene+expression+circuit.">In silico feedback for in vivo regulation of a gene expression circuit.</a> Nat. biotechnol. 29, 1114-1116 (2011).</li></ol>

McIsaac, Noyes, and Botstein (with others) have submitted part of this system as a Patent Disclosure.

The hormone-based switches described here have myriad applications, from studying native cell biology to testing the ability of a DNA binding domain of interest to target a DNA sequence in vivo. The system has many desirable features, including a graded response to inducer, fast action, and tight regulation (i.e.&#160;no measurable leakiness, see Figure 7). However, there is still room for improvement and expansion.
Recent work in synthetic biology has emphasized multiplexing synthetic systems and expanding the repertoire of well-characterized, programmable DNA parts16. Independent, conditional expression of distinct target genes requires both multiple inducers and DNA binding domains that lack overlapping specificity. The availability of engineered and naturally occurring receptors provides a large set of parts with which to develop new artificial transcription factors.&#160;Expanding the repertoire of mutually orthogonal switches has been greatly aided by directed evolution approaches17,18. Current efforts to reprogram the ligand-binding specificity of our artificial transcription factors will be presented in the future.
Previously, the phenotypic consequences of gene deletion/overexpression have been extensively studied in yeast19,20. However, it has not been straightforward to study the effect of expression on different phenotypes over a range of expression levels. A primary feature of the system presented herein is its graded nature (Figure 8). While often assumed, the relationship between gene expression and phenotypes of interest may not necessarily be monotonic. Artificial transcription factors such as Z3EV and Z4EV will make the task of assaying the phenotypic responses to changes in gene expression straightforward.
Finally, the protocols discussed in this manuscript are for engineering and characterizing artificial transcription factors that respond to &#946;-estradiol; however, an important alternative to chemically responsive domains is the use of light-responsive domains (such as the PhyB/Pif3, LOV, and BLUF), whose activities are reversible without having to perturb the culture growth medium21-23. Light-based systems facilitate spatiotemporal control of gene expression, protein-protein interactions, ion transport, and enzymatic activity, which have already proven powerful21-26.
In conclusion, we have presented methods for the rapid construction of inducible, artificial transcription factors in yeast. This protocol provides a template for their construction in conjunction with cognate GFP reporters, as well as methods for analyzing the reporter data using flow cytometry and assaying the effect of ATF activation on growth.

Developing genetic switches in yeast is of great interest in both academic and industrial research. In the ideal case, genetic switches can be used to activate a particular gene (or set of genes) only when desired by the experimenter. A gene&#39;s coding sequence placed downstream of a synthetic promoter is not expressed in the absence of an inducing molecule: upon inducer addition the gene should be rapidly expressed. It has only recently been demonstrated that such a switch can be engineered in yeast, where in the absence of inducer, the strain displays a deletion phenotype, but in the presence of inducer, expression is activated in proportion to the level of inducer in all cells1,2. Historically, conditional expression systems in yeast have relied heavily upon nutrient-responsive DNA sequences, including&#160;the&#160;MET, PHO, and GAL promoters (whose activities are sensitive to extracellular levels of methionine, phosphate, and galactose, respectively). While conditional expression can be achieved, it comes at the cost of significant pleiotropic effects.
Hormone receptors such as the human estrogen receptors have proven to be effective switches in eukaryotes3,4. In the absence of hormone, a protein fused to a hormone receptor can be sequestered in the cytoplasm where it interacts with the Hsp90 chaperone complex. Upon binding the appropriate ligand, the receptor undergoes a conformational change, causing it to be released from the Hsp90 chaperone complex and revealing a nuclear localization signal. To observe the localization dynamics of a particular hormone receptor-containing protein, we created a C-terminal fusion of Gal4dbd.ER.VP16 (GEV, a synthetic transcription factor containing the Gal4p DNA binding domain, the ligand binding domain of the human estrogen receptor, and VP16) with GFP2. Nuclear localization was observed within 8 min following addition of saturating amounts of the inducer, &#223;-estradiol, to which wild type yeast is completely inert. By this time, the majority of cells have clearly visible active transcription sites for GEV target genes (assayed via fluorescence in situ hybridization) as well as fully mature mRNAs2,5. GEV target genes increased in expression &#62;2-fold within 2.5 min2,6 (measured using microarrays), demonstrating that it takes &#60;2.5 min&#160;for the chimeric activator to translocate to the nucleus, bind DNA, and activate transcription.
While several other on-switches have been applied in yeast that utilize various small molecules or drugs (including the classic doxycycline-based switches from Escherichia coli7,8 and an indigo-based switch9 from Arabidopsis thaliana), none have achieved the speed, specificity, or tightness of regulation exhibited by the hormone-based switches. It is worth mentioning that rapid off-switches have also been developed in yeast, and typically work by fusing a gene to a protease or ubiquitin ligase targeting sequence2,10,11,12. The ability to rapidly remove a target protein from the cell facilitates the study of essential genes as well as genes that are prone to genetic suppression when deleted10.
The methods described in this protocol are for building and characterizing synthetic on-switches in yeast. First, we show how to rapidly generate genomically-integrated fusion proteins consisting of a DNA binding domain of interest, the ligand binding domain of the human estrogen receptor, and VP16 (DBD-EV&#8217;s). Following our protocol, the fusion protein is expressed from an ACT1 promoter. We then show how to create a cognate reporter plasmid for the ATF and test its functionality using flow cytometry. Though we envision these reporter plasmids being used with new ATFs (Figure 1), they can be used as reporters for a native transcriptional activator as well. Finally, details for testing the effect of ATF activation on cell growth in 96-well plates and for engineering inducible genomic alleles are provided.

<table border="0" cellpadding="0" cellspacing="0" width="352">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Name of the Reagent</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:88px;">Comments&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:88px;">Expand High Fidelity PCR System</td>
			<td style="width:88px;">Roche</td>
			<td style="width:88px;">11 732 650 001</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">T4 DNA Ligase</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">M0202S</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Competent E. coli</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">18258-012</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Estradiol</td>
			<td style="width:88px;">Tocris Biosciences</td>
			<td style="width:88px;">2824</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Ampicillin</td>
			<td style="width:88px;">Fisher Scientific</td>
			<td style="width:88px;">BP1760-5</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:88px;">T4 Polynucleotide Kinase</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">M0201</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">PBS</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">10010-23</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">Tween-20</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">9005-64-5</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">clonNAT</td>
			<td style="width:88px;">Jena Bioscience</td>
			<td style="width:88px;">AB-102L</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">XbaI</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">R0145S</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">NotI-HF</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">R3189S</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">CutSmart Buffer</td>
			<td style="width:88px;">NEB</td>
			<td style="width:88px;">B7204S</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Glucose</td>
			<td style="width:88px;">Fisher Scientific</td>
			<td style="width:88px;">D15-500</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Bacto-Peptone</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">211677</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Yeast Extract</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">210929</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Agarose</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">214010</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">100% Ethanol</td>
			<td style="width:88px;">Decon Laboratories</td>
			<td style="width:88px;">04-355-222</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">G418</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">11811-031</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:88px;">QIAGEN</td>
			<td style="width:88px;">27104</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:88px;">Lithium acetate dihydrate</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">L-6883</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:88px;">Salmon sperm DNA</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">93283</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">PEG 3350</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">P-3640</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:88px;">Plasmids pMN8 and pMN10; yeast strain yMN15</td>
			<td style="width:88px;">Noyes or Botstein labs</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Luria Broth</td>
			<td>Sigma-Aldrich</td>
			<td>L3397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Material Name</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalogue Number</td>
			<td style="width:88px;">Comments (optional)</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">LSRII Flow Cyometer&#160;</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">Various Models</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="103">
			<td height="103" style="height:103px;width:88px;">MATLAB or FlowJo</td>
			<td style="width:88px;">MATLAB or FlowJo</td>
			<td style="width:88px;"></td>
			<td style="width:88px;">Software for analyzing .FCS files from flow cytometry experiments</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:88px;">R</td>
			<td style="width:88px;">Open Source</td>
			<td style="width:88px;"></td>
			<td style="width:88px;">For analyzing growth-rate data from plate reader.&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Falcon Tubes</td>
			<td style="width:88px;">BD Biosciences</td>
			<td style="width:88px;">352052</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:88px;">1.7 ml Eppendorf&#160; Tubes</td>
			<td style="width:88px;">GeneMate</td>
			<td style="width:88px;">C3262-1</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">250 ml Shake Flasks</td>
			<td style="width:88px;">Corning</td>
			<td style="width:88px;">4446-250</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">96-well, flat-bottom plates</td>
			<td style="width:88px;">Costar</td>
			<td style="width:88px;">3635</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:88px;">Plate Reader (Synergy H1 Multi-Mode Plate Reader)</td>
			<td style="width:88px;">BioTek</td>
			<td style="width:88px;">H1MG</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Breathe-Easy Membranes</td>
			<td style="width:88px;">Sigma-Aldrich</td>
			<td style="width:88px;">Z380059-1PAK</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">Thermocycler</td>
			<td style="width:88px;">various companies</td>
			<td style="width:88px;">Various models</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">SC-Ura powder</td>
			<td style="width:88px;">MP Biomedicals</td>
			<td style="width:88px;">114410622</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:88px;">5-FOA</td>
			<td style="width:88px;">Zymo Research</td>
			<td style="width:88px;">F9001-1</td>
			<td style="width:88px;"></td>
		</tr>
	</tbody>
</table>

rapid synthesis and screening of chemically activated transcription factors with gfp-based reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

Figure 5 shows induction of GFP by Z3EV, Z4EV, or GEV over time. Note the increase in GFP production in response to the ATFs containing nonyeast DNA binding domains. We recently speculated that this results from these factors achieving single-gene specificity in the yeast genome1. GEV induction alone leads to activation/repression of hundreds of genes, whereas Z3EV and Z4EV only activate expression of a target gene placed downstream of a synthetic promoter containing appropriate binding sites (5&#39;-GCGTGGGCG-3&#39; and 5&#39;-GCGGCGGAGGAG-3&#39;, respectively)1. Figure 7 demonstrates the tight regulation of the system (no inducer results in no detectable GFP) and that all cells get induced in response to inducer&#160;The ability of Z3EV to induce GFP across a range of &#946;-estradiol concentrations is shown in Figure 8.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig1highres.jpg" src="/files/ftp_upload/51153/51153fig1.jpg" /> 
Figure 1. Artificial transcription factors interact with Hsp90 in the absence of &#946;-estradiol. When &#946;-estradiol binds to the ATF, Hsp90 dissociates, allowing the ATF to enter the nucleus and activate expression from the plasmid reporter.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig2highres.jpg" src="/files/ftp_upload/51153/51153fig2.jpg" /> 
Figure 2. Schematic of protocol for engineering strains that contain artificial transcription factors and building/testing cognate GFP reporters.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig3highres.jpg" src="/files/ftp_upload/51153/51153fig3.jpg" /> 
Figure 3. The ACT1pr-URA3-EV sequence in the strain yMN15 at the LEU2 locus.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig4highres.jpg" src="/files/ftp_upload/51153/51153fig4.jpg" /> 
Figure 4. Design of PCR primers for amplifying a DNA binding domain of interest with additional sequence homology to generate a new fusion protein when successfully transformed into yMN15.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig5highres.jpg" src="/files/ftp_upload/51153/51153fig5.jpg" /> 
Figure 5. GFP induction by three different ATF-promoter pairs in response to 1 &#181;M &#946;-estradiol.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig6highres.jpg" src="/files/ftp_upload/51153/51153fig6.jpg" /> 
Figure 6. A MATLAB script for processing flow cytometry data. This script was adapted from&#160;<a href="http://openwetware.org/wiki/McClean:_Matlab_Code_for_Analyzing_FACS_Data">http://openwetware.org/wiki/McClean:_Matlab_Code_for_Analyzing_FACS_Data</a>.
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig7highres.jpg" src="/files/ftp_upload/51153/51153fig7.jpg" /> 
Figure 7. Induction of GFP by Z3EV in response to 0 &#181;M &#946;-estradiol and 1 &#181;M &#946;-estradiol following 18 hr of induction. Fluorescence was also measured from cells lacking GFP to illustrate the tight regulation of the Z3EV system. This figure was generated using the code in Figure 6.
<img alt="Figure 8" fo:content-width="5in" fo:src="/files/ftp_upload/51153/51153fig8highres.jpg" src="/files/ftp_upload/51153/51153fig8.jpg" /> 
Figure 8. The relationship between inducer concentration and expression output is graded with a Hill coefficient of ~1. (A) Distributions&#8203; of GFP expression as a function of &#946;-estradiol concentration.&#160;(B) The mean fluorescence of the distributions from (A). The data were fit to the function G(D) = Gmin + (Gmax&#8722;Gmin)Dn/(Kn+Dn) where D is the &#946;-estradiol dose, Gmin and Gmax are the minimum and maximum GFP values, respectively, n is the Hill coefficient, and K is the &#946;-estradiol dose that yields &#189; (Gmax + Gmin).
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always" width="176">
	<tbody>
		<tr>
			<td>Oligonucleotide Sequence</td>
			<td>Name</td>
		</tr>
		<tr>
			<td>5&#8217;-TTGAAACCA 
			AACTCGCCTCT-3&#8217;</td>
			<td>DBD Check Forward</td>
		</tr>
		<tr>
			<td>5&#8217;-tccagagac 
			ttcagggtgct-3&#8217;</td>
			<td>DBD Check Reverse</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Primers for confirming correct fusion of DBD of interest to the Estrogen Receptor. The forward primer is homologous the ACT1 promoter and the reverse primer is homologous to the estrogen receptor. For Typical DBDs (~300 bp), the PCR product is &#60;1.5 kb.

<table border="0" cellpadding="0" cellspacing="0" width="176">
	<tbody>
		<tr>
			<td>Oligonucleotide Sequence</td>
			<td>Name</td>
		</tr>
		<tr>
			<td>5&#39;-ggccgc&#8230;DBD binging site... t-3&#39;</td>
			<td>Top oligo</td>
		</tr>
		<tr>
			<td>5&#8217;-ctaga&#8230;DNA binding site reverse&#8230;gc-3&#8217;&#160;&#160;</td>
			<td>Bottom oligo</td>
		</tr>
		<tr>
			<td>5&#39;-gccgcGTGGGCGTGCGTGGGCGGG 
			CGTGGGCGTGCGTGGGCGGGCGTG 
			GGCGTGCGTGGGCGt-3&#39;</td>
			<td>Top oligo + 6x Zif268 Binding Sites</td>
		</tr>
		<tr>
			<td>5&#8217;-ctagaCGCCCACGCACGCCCACGCC 
			CGCCCACGCACGCCCACGCCCGCC 
			CACGCACGCCCACgc-3&#8217;&#160;</td>
			<td>Bottom oligo + 6x Zif268 Binding Sites</td>
		</tr>
	</tbody>
</table>

Table 2.&#160;Structure of primers for creating dsDNA containing binding sites of interest. To create the Z3EV-responsive promoter, the oligonucleotides Top oligo + 6x Zif268 Binding Sites and Bottom oligo + 6x Zif268 Binding were used. Sequences for creating the correct DNA overhangs are in lowercase. The Zif268 consensus binding site, GCGTGGGCG, is underlined in the top oligo.

<table border="0" cellpadding="0" cellspacing="0" width="176">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase Buffer</td>
			<td>5 &#181;l</td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase (@10,000 units/ml)</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>Top oligo (100 &#181;M)</td>
			<td>1.5 &#181;l</td>
		</tr>
		<tr>
			<td>Bottom oligo (100 &#181;M)</td>
			<td>1.5 &#181;l</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>41 &#181;l</td>
		</tr>
	</tbody>
</table>

Table 3.&#160;Phosphorylate and anneal primers in a thermocycler using 50 &#181;l&#160;of reaction described above. There are two thermocycler steps. Step 1:&#160;37 &#176;C 30 min&#160;(Phosphorylate), and step&#160;2:&#160;95 &#176;C 30 sec&#160;(step down 1 &#176;C every 30 sec&#160;until at 4 &#176;C; Anneal).

<table border="0" cellpadding="0" cellspacing="0" width="176">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>XbaI</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>NotI-HF</td>
			<td>1 &#181;l</td>
		</tr>
		<tr>
			<td>Plasmid DNA</td>
			<td>5 &#181;l (1-2 &#181;g)</td>
		</tr>
		<tr>
			<td>10x Cut Smart Buffer</td>
			<td>5 &#181;l</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>38 &#181;l</td>
		</tr>
	</tbody>
</table>

Table 4.&#160;Restriction digest of plasmid pMN8 with XbaI and NotI.

<table border="0" cellpadding="0" cellspacing="0" width="176">
	<tbody>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Reagent</td>
			<td class="xl66" style="width:66pt" width="88">Amount</td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td>T4 Ligase Buffer</td>
			<td>2 &#181;l</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td>T4 Ligase</td>
			<td>0.2 &#181;l</td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td>Backbone @ 10 ng/&#181;l</td>
			<td>1 &#181;l</td>
		</tr>
		<tr height="106" style="height:79.5pt">
			<td>Binding Sequences@ 5x molar concentration/&#181;l</td>
			<td>1 &#181;l</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td>Water</td>
			<td>15.8 &#181;l</td>
		</tr>
	</tbody>
</table>

Table 5.&#160;Ligate binding sites into plasmid DNA.

<table border="0" cellpadding="0" cellspacing="0" width="176">
	<tbody>
		<tr>
			<td>Primer</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>~40 bp upstream of ATG + CGCACTTAACTTCGCATCTG-3&#8217;</td>
			<td>Forward primer for amplifying KanMX-Promoter</td>
		</tr>
		<tr>
			<td>5&#8217;- reverse complement(ATG + ~37bp) + TATAGTTTTTTCTCCTTGACG-3&#8217;</td>
			<td>Reverse primer for amplifying KanMX-Promoter</td>
		</tr>
		<tr>
			<td>5&#8217;-caatttgtctgctcaagaaaataaa 
			ttaaatacaaataaaCGCAC 
			TTAACTTCGCATCTG-3&#8217;</td>
			<td>Forward primer for making GCN4 promoter swap.</td>
		</tr>
		<tr>
			<td>5&#8217;-tggatttaaagcaaataaacttgg 
			ctgatattcggacatTATAGTT 
			TTTTCTCCTTGACG-3&#8217;</td>
			<td>Reverse primer for making GCN4 promoter swap.</td>
		</tr>
	</tbody>
</table>

Table 6.&#160;PCR amplify KanMX-Promoter for making titratible allele.

Watch this Scientific Journal Video about Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters at JoVE.com

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

rapid-synthesis-screening-chemically-activated-transcription-factors

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n- (E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6 Notable milestones are the oxidative coupling of Ge94- clusters to oligomers and infinite chains,7-19 their metallation,14-16,20-25 capping by transition-metal organometallic fragments,26-34 insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47 addition of main-group organometallic fragments as exo-bonded substituents,48-50 and functionalization with various organic residues by reactions with organic halides and alkynes.51-58 
This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4Ge9 from which the Ge94- clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4Ge9 is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3SiC&equiv;CSiMe3. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2C=CH-Ge9-CH=CH2]2-. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en which were characterized by single-crystal X-ray diffraction. 
The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.

We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...