Final Report Summary - BIOASSAYDEVICE (A device for biological high-throughput assays based on in vitro compartmentalisation) We have developed a microfluidic system that can simultaneously monitor the time-dependence of co-induced protein expression of monomeric red fluorescent protein (mRFP1) and the enzymatic activity of alkaline phosphatase (AP) in compartmentalized E.coli cells. To demonstrate the utility of this methodology for applications in directed evolution, wild type AP (WT AP) was analyzed against its less active mutant (R166S AP). The device was designed to be able to store up to 4,000 droplets in storage wells. Also, the droplet shrinking due to water diffusion into the PDMS matrix that jeopardizes quantitative measurements of droplet contents was resolved by integrating a reservoir underneath the wells to continuously supply water to the stored droplets to keep the water content constant over long period.The significant differences in expression rates of mRFP1 between droplets are primarily due to different numbers of cells in droplets as a consequence of Poissonian encapsulation of cells (Figure 1A). This interpretation is backed up by the observation of distinctive groups that provide three regularly-spaced and well-defined peaks in the histogram of mRFP1 production, which correspond to the initial occupancy of zero, one or two cells per droplet. There is significant intra group variation (0.45 ~ 1.55 millions copies of mRFP1 per droplet) observed in the first group that started with one cell. The time courses for fluorescein formation in droplets are shown in Figure 1B. The rates observed for individual droplets fall into three distinct groups as observed for mRFP1 expression, again reflecting the number of cells initially compartmentalized. For directed evolution experiments it is crucial to be able to distinguish between different enzyme variants. The microfluidic platform was thus used to differentiate the activities of WT AP from its less-active mutant R166S AP having a smaller kcat/Km as shown in Figure 1C. The normalization correlates mRFP1 and AP expression levels, and removes the variance introduced by different copy numbers of plasmid DNA or different numbers of cells in droplets. Gaussian fits in (Fig. 3a) can be ascribed to fluctuations of the relative expression level of AP and mRFP1 despite being under the control of the same promoter. Mutant and wild-type enzyme can be distinguished, but there is a considerable overlap (~50% of the respective populations) despite a large difference in kcat/KM. By constructing a plot correlating mRFP1 and fluorescein production rate (Figure 1D), populations of WT AP and R166S become now clearly distinguishable. The linearity of each series implies that mRFP1 expression is positively correlated with fluorescein production and hence AP expression. By using the microfluidic system described in this work, it was possible to maintain thousands of droplets in a constant environment that allows quantitative measurements of each droplet and enabled the concurrent study of the kinetics of protein expression and enzymatic activity in individual cells. The ability to simultaneously monitor these properties provides an analytical tool for the assessment of members of a library in a directed evolution experiment or allows interrogation of the heterogeneity of cells generated from an identically prepared ensemble. Documentos relacionados final1-220946-772446-junguk-final-report.pdf
