Our protocol makes it easier and faster to understand relationships between biological parts. We can test the behavior of many ratio metric combinations of parts in a single well. This technique allows researchers to characterize the complex relationships between circuit components more comprehensively with greater experimental efficiency than conventional approaches.
This method is applicable for synthetic biology and for any biological questions, probing behavioral changes in a cell in response to different levels of transfected genes where the output can be measured via flow cytometry. Begin preparing tubes for each DNA aggregate by setting aside 1.5-milliliter micro centrifuge tubes labeled as no color control, mKO2 control, TagBFP control, neon green control, all color control, poly-transfection mix one and poly-transfection mix two. Add 36 microliters of reduced serum medium to the no color control, mKO2 control, TagBFP control, neon green control and all color control tubes, then add 18 microliters of reduced serum medium to each poly-transfection mix one and poly-transfection mix two tubes.
Next, add 600 nanograms of filler plasmid to the no color control tube, 300 nanograms of mKO2 and 300 nanograms of filler plasmid to the mKO2 color control tube and add 300 nanograms of TagBFP and 300 nanograms of filler plasmid to the TagBFP color control tube. To the neon green color control tube, add 300 nanograms of constitutive neon green and 300 nanograms of filler plasmid, then add 100 nanograms of each mKO2, TagBFP, constitutive neon green and 300 nanograms of filler plasmid to the all color control tube. To the poly-transfection mix one tube, add 150 nanograms of mKO2, 75 nanograms of reporter neon green plasmid and 75 nanograms of filler plasmid.
To the poly-transfection mix two tube, add 150 nanograms of TagBFP, 75 nanograms of L7Ae plasmid and 75 nanograms of filler plasmid. Once done, create the transfection master mix in a 1.5-milliliter micro centrifuge tube by combining 216 microliters of reduced serum medium with 9.48 microliters of transfection reagent. Mix well by pipetting up and down before setting it aside.
Add 1.58 microliters of enhancer reagent to no color control, single color control and all color control tubes and add 79 microliters of enhancer reagent to each poly-transfection mix tubes. Mix each tube individually by pipetting vigorously. Add 37.58 microliters of transfection master mix to the no color control, single color control and all color control tubes and mix by pipetting vigorously.
Next, add 18.79 microliters of transfection master mix to each poly-transfection mix tubes and mix each tube well with vigorous pipetting. Dispense the transfection mixes into the wells by pipetting 65.97 microliters of each transfection mix for the no color, single color and all color controls into the corresponding wells, then transfer 32.98 microliters of the poly-transfection mix one into the poly-transfection well and distribute the complexes effectively by swirling the plate quickly but gently in a tight, figurate pattern along a flat surface, then pipette 32.98 microliters of the poly-transfection mix two into the same poly-transfection well and swirl the plate in the same fashion. Incubate cells for two days.
Perform flow cytometry to read out the fluorescence of the transfection markers and output reporter for each cell. A well-performed co-transfection, distinct from the poly-transfection shown in the video, shows a tight correlation between TagBFP and EYFP, expressing plasmids that were co-delivered. In contrast, a poorly performed co-transfection shows a poor correlation between the two plasmids.
Well-performed poly-transfection showed good coverage of the two-dimensional space and good compensation of any spectral bleed through between fluorescent proteins. Poly-transfection data with a low number of live cells was difficult to subdivide into enough bins with a sufficient number of cells in each bin for analysis. A poly-transfection with poor transfection efficiency resulted in sparse coverage of the two-dimensional space for analysis.
The effective DNA ratio on output expression was tested by placing a repressor protein, L7Ae with mKO2, and an output protein, neon green with TagBFP, in separate transfection mixes. Neon green expression was then monitored at different levels of mKO2 and TagBFP. Co-transfection and poly-transfection results were compared for this circuit.
Results of 11 individual co-transfections that combined the DNA parts shown previously were collated into a single dataset and compared to the poly-transfection data. Comparison of the dose response curves of L7Ae repressing the output reporter showed that the median output level per bin was very similar between the co-transfection and poly-transfection data. A successful application of poly-transfection was demonstrated for optimizing a cell type classifier.
The micro RNA-21 present in HeLa cells and not-HEK cells acts to knock down the expression of BM3R1 because BM3R1 represses production of the circuit output mKO2. When micro RNA 21 is present in a cell, mKO2 expression will be turned on. The amount of Gal4-VP16 tunes mKO2 expression at low levels of BM3R1.
Each circuit component was co-delivered with a different fluorescent marker. This poly-transfection quantifies how much of each circuit component each cell received. mKO2 is produced in MIR21, producing HeLa cells but not in HEK cells.
mKO2 output was analyzed after delivering these circuit components in separate transfection mixes with distinct fluorescent reporters to indicate the amount of each circuit component received by a particular cell. This sub-sampling predicted that at this ratio, the co-transfected circuit had a 91%specificity, 62%sensitivity and 77%accuracy, when classifying HEK293 versus HeLa cells. The most common pain point in this protocol is a lack of a tight correlation within each transfection mix.
So remember to vigorously mix the transfection tubes right after adding the enhancer reagent and then be gentle with mixing and pipetting the transfection tubes after the lipid containing transfection mix is added The genetic components in this procedure could be replaced with any other genetic components to answer questions about their functioning performance in various environments. This method has rapidly developed genetic circuits, such as cell classifiers, feedback and feedforward controllers, bi-stable motifs and many more.
