Team:BostonU/Measurement


Overview

BostonU has characterized two light inducible promoters in yeast across pulsing and saturating light programs by leveraging the eVOLVER, a novel culturing platform. The eVOLVER offers improved scale over traditional culturing methods by separating experiments across 16 vials, each with individually controlled temperature, optical density, dilution rate, and tunable light programs. Developed in Boston University’s Khalil Lab, the eVOLVER enables sampling of a wide parameter space in optimization of use of these new tools. BostonU is the first group to fully probe eVOLVER light programming capabilities, measuring reporter expression from S. cerevisiae strains expressing the light inducible promoters LOV2 (blue light) and PhiReX (red light). Improved scalability allowed testing of multiple iterations of transcription schemes with promoter and phytochrome expression cloned across selection markers and compared for peak performance. Reporter expression from optimal strains was characterized across light programs, elucidating the performance of these new tools for tunable transcriptional control.

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Running an experiment

While the protocols for sampling cultures during an experiment in the eVOLVER and with flasks or blocks grown in the shaking incubator are similar, the eVOLVER offers better control of samples through automatically keeping optical density constant or turning on and off LEDs to activate light inducible systems. Both methods require the manual sampling of the culture (pipetting a portion of the sample out and fixing the cells), however using a block would also require you to manually dilute samples to retain a near constant optical density. Running an experiment on light inducible systems becomes more complicated when using a block because light sources must be manually turned on and off at the correct times. In contrast, the eVOLVER will automatically dilute cultures so that the OD is within a range that the user sets as well as automatically control which colored LEDs are on based on the user's program. The eVOLVER offers more efficient experiments, allowing you to control each individual culture as well. In the shaking incubator in a culture block, it is difficult to target light at just one well. The eVOLVER, however, can control each vial separately, allowing for more types of experiments to be run in an efficient way.

eVOLVER's Control

We put our no construct yeast strain, yPH500, as well as our LOV2 yeast strain in the eVOLVER and in the shaking incubator. The shaking incubator requires the user to manually dilute the cultures, which creates more room for error. Although we tried our best, diluting the cultures by hand in a culture block did not offer great control over our experiment. Here are two instances when we put our no construct yeast strain into the eVOLVER:

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Figure 1: No construct yeast strain in the shaking incubator

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Figure 2: LOV2 yeast strain in the shaking incubator

We see in these two examples that, while the cell count can remain fairly constant in the shaking incubator, it is not reliable in keeping the cell count constant.

In contrast, the yeast cultures in the eVOLVER have controlled ODs, remaining at a steady cell count because of the eVOLVER's automatic dilutions:

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Figure 3: No construct yeast strain in the eVOLVER

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Figure 4: LOV2 yeast strain in the eVOLVER

Improved Data Using the eVOLVER

Using the eVOLVER, we have seen better, more reliable data for characterizing our yeast strains:

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Figure 5: LOV2 (blue) and no construct (yellow) yeast strains in the shaking incubator

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Figure 6: LOV2 (blue) and no construct (yellow) yeast strains in the eVOLVER

We see that LOV2 is able to express more in the eVOLVER. In addition, since our strains have an adenine mutation that causes a pink color in our strains, diluting the cultures to prevent pigment buildup is important in ensuring that our mRuby (red) fluorescence measurements are valid. With the eVOLVER's automatic and precise diluting capabilities, we see in Figure 6 that the eVOLVER prevents buildup of pigment in our no construct strain, while the shaking incubator data in Figure 5, we see that there is some fluorescence, presumably from the buildup of the pink color of our yeast. Using the eVOLVER, we are able to better control our experiments as well as obtain better data.

While running an experiment, the eVOLVER collects data from each of the 16 vials and displays it on a user interface. This allows the user to track how each of their cultures are doing in real time in terms of optical density and temperature. In contrast, the culture block method requires guesswork during the experiment. In fact, the eVOLVER lets the user download this OD data as well, which we have used to create plots of OD vs time from our blue light step experiment:

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Figure 7: OD data received from the eVOLVER, which is also displayed real-time when running an eVOLVER experiment

The eVOLVER is able to give us real-time quantitative information on our yeast cultures while running experiments as well as better control our yeast cultures through automation. Using the eVOLVER enables us to get more reliable data and characterize our systems more precisely. Being able to improve the control over our systems provides greater confidence in our experimental results.