The overall goal of this procedure is to create, test and validate genetically encoded, inducible artificial transcription factors or ATFs with desired specificity. This is accomplished by first creating the A TF via a simple yeast transformation in which inclusion of A DNA binding domain PCR product results in an in-frame fusion with a human estrogen receptor and VP 16. The second step of the procedure is to create a GFP reporter plasmid for the A TF by replacing the binding sites in the plasmid PMN eight with a TF targeting sequences.
The third step of the procedure is to assay the ATFs ability to activate the GFP reporter and to also assess its effect on yeast physiology by measuring the growth rate. The final step of the procedure is to use the validated a TF to conditionally express, a native genomic target. Ultimately, any target gene of interest can be made conditionally active.
The main advantage of this technique over existing methods like galactose mediated induction of gene expression, is that selective induction can be achieved under diverse nutrient and physiological conditions without pleotropic effects. This method can help answer key questions in the yeast biology through the fast and reversible introduction of desired gene products, enabling study of both gain and loss of function. To begin, PCR amplify the DNA binding domain or DVD of interest using a high fidelity polymerase.
Transform greater than one microgram of the purified PCR product into yeast using the lithium acetate method. Following transformation, spin the cells at top speed for 15 seconds in a micro centrifuge, remove the transformation mix and resuspend the cells in approximately 200 microliters of sterile water before plating on yeast extract. Pepto dextrose or YPD medium the next day replica plate from YPD onto five fluoro erotic acid plates.
After growth for two to four days, rere at least five to 10 colonies onto YPD. Then perform a colony PCR using the primers in the text protocol and sequence to verify inclusion. Dilute the desired binding region oligos to equimolar concentrations.
Phosphorylate the binding region using T four polynucleotide kinase, and then a neo thermocycler according to the reaction conditions in the text protocol. Next, digest approximately one to two micrograms of PMN eight with not one HF and xb. One for one hour at 37 degrees Celsius gel.
Purify the backbone band and dilute the purified backbone to 10 nanograms per microliter. Then dilute the double stranded phosphorylated binding site to five times the molar concentration of backbone per microliter. Using T four DNA Ligase ligate the binding sites into the digested backbone at room temperature for 30 minutes.
Perform the transformation by adding half the ligation reaction to 50 microliters of standard chemically competent EIA coli cells, and following the procedure listed in the text protocol. After recovering the cells, add 100 to 200 microliters of cells per luria berti or lb plus ampicillin plate, an incubate overnight. The next day grow up e coli and lb plus ampicillin liquid and harvest the plasmid DNA as described in the text protocol.
Then sequence verify the new reporter plasmid from ligation colonies. Finally transform the new reporter plasmid into the A TF containing E strain using lithium acetate transformation. To begin grow a TF plus reporter containing strain overnight in five milliliters of synthetic complete medium lacking uracil.
Obtain 9 250 milliliters, shake flask, and add 25 milliliters of SC minus URA to each. Then prepare seven of the flasks with different dilution of beta estradiol as listed in the text protocol from tenfold serial dilution of a 25 millimolar beta estradiol stock at 125 microliters of the overnight cultures to these seven flask for the two remaining flasks inoculate with a constitutive GFP producing strain and a strain lacking GFP. These are needed for calibrating the flow cytometer.
Shake the flask at 200 RPM and 30 degrees Celsius for 12 to 18 hours. Then remove 500 microliters of each culture and spin down in separate 1.7 milliliter einor tubes. After aspirating the supernatant, rinse the pelleted cells once with flow cytometry buffer, then resuspend the cells in one milliliter of the same buffer.
Next, add one milliliter of flow cytometry buffer to nine falcon two. Add the resuspended cells to the buffer containing falcon tubes for a final volume equal to two milliliters. Finally, perform flow cytometry.
Use forward scatter and side scatter to identify yeast cells. Set the flow rate to approximately 3000 cells per second. Measure the GFP through the 50 channel and proceed as in the text protocol from frozen stocks streak out both an A TF containing strain and an A TF lacking strain onto YPD After two days of growth, inoculate separate culture tubes containing five milliliters of YPD liquid and grow each strain overnight at 30 degrees Celsius.
Perform tenfold serial dilutions of a 0.25 millimolar beta estradiol stock solution up to 10, 000, fold in 100%ethanol. Add 40 microliters of each overnight culture to 10 milliliters of YPD liquid. For each strain, add 96 microliters of the diluted cells to 18 wells of a 96 well plate.
Then add four microliters of beta estradiol to the cells. An essential point is to make sure each well has the same amount of total ethanol. Perform a triplicate with three of the wells for each strain being ethanol only.
Controls cover the 96 well plate in a gas permeable membrane. Monitor the growth at an optical density of 600 nanometers in a plate reader. Quantify the growth rates using any number of methods, such as finding the maximum slope.
After preparing the plasmid as instructed in the text protocol PCR, amplify the can MX promoter cassette. Transform the PCR product into an A TF expressing strain using the lithium acetate method plate, the transform cells onto YPD and replica plate onto YPD plus G four 18 antibiotic the following day. Finally confirm the appropriate insertion of the promoter using the forward primer and a reverse primer internal to the gene whose promoter has been replaced.
The final PCR product is approximately 1.2 KB shown here is the induction of GFP by three different a TF promoter pairs Z four EVZ three EV, or GEV over time in response to one micromolar beta estradiol. Note the increase in GFP production in response to the ATFs containing non yeast DNA binding domains. This may result from these factors achieving single gene specificity in the yeast genome.
GEV induction alone leads to activation or repression of hundreds of genes, whereas Z three EV and Z four EV only activate expression of a target gene placed downstream of a synthetic promoter containing appropriate binding sites. Here, induction of GFP by Z three EV in response to zero nanomolar beta estradiol, and one micromolar beta estradiol following 18 hours of induction is shown Fluorescence was also measured from cells lacking GFP to illustrate the tight regulation of the Z three EV system. The ability of Z three EV to induce GFP across a range of beta estradiol concentrations is shown here.
The mean fluorescence of the distributions reveals that the relationship between inducer concentration and expression output is graded with a hill coefficient of approximately one indicating independent binding of Z three EV Following this procedure, other methods like gene expression microarrays could be performed in order to answer additional questions like how changing the expression of a single gene affects genome wine patterns of gene expression. After watching this video, you should have a good understanding of how to engineer, test and validate the efficacy of artificial transcription factors. These artificial transcription factors conditionally control expression of genes placed downstream of appropriate DNA target sequences.
