High-resolution FACS experiments can sort cells from a population only to the extent that the fluorescence level of the cell correlates with the phenotype that is being examined. In trying to develop a staining protocol that yields good correlation between fluorescence and, in this case, PHB levels, important factors are (i) promoting specific fluorescent molecule binding to the target (PHB) and (ii) allowing adequate access of the fluorescent molecule to the target.
In the Nile red optimization for PHB fluorescence, molecule specificity was addressed by varying the environment for staining. This was especially important in Synechocystis. Synechocystis has multiple layers of thylakoid membrane, which is used in its photosynthetic apparatus. These membranes provide a large area of lipid-like interfaces for nonspecific binding of Nile red. To minimize binding of the Nile red to the thylakoid membrane, the ionic strength of the medium and the dye concentration were examined. When the ionic strength of the staining environment was changed from a sodium chloride solution to deionized water, the Nile red stained the PHB more specifically. Additionally, the dye concentration also strongly affected the resolution of the assay. In E. coli, this was not as important. The effect of dye concentration increased, unlike in Synechocystis, where a maximum was observed. The ionic strength did not affect the resolution in E. coli (data not shown). These observations can be attributed to the lack of large membrane structures in E. coli.
Providing consistent access of the fluorescent molecule to the target is also necessary for determining a quantitative fluorescence level. In Synechocystis, the stain readily permeated the cell and stained the PHB granules. Figure shows that even in the prior staining protocol, all Synechocystis cells were being stained. This should be contrasted with Fig. , which shows that a large portion of the E. coli cells did not stain at all for PHB in the nonoptimized protocol. Synechocystis is a naturally competent cell and as such is able to take up DNA molecules readily. This may imply that the morphology of the Synechocystis membrane may allow it to take up Nile red more readily than E. coli, which is not naturally competent. To improve the dye transport across the E. coli cell membrane, competent-cell protocols and other permeabilization methods were attempted. Of these, sucrose shock permeabilized the cells in such a way that the Nile red could enter the cytoplasm and stain the granule.
While the E. coli cells could now take up the Nile red, most of the cells were killed in the process. Further optimization was required to increase the cell viability while retaining good staining properties. Adjusting the sucrose concentration and the buffers used improved the viability to 48%. This will allow an adequate efficiency for screening mutant libraries by FACS.
To validate the use of resolution (equation 1
) as a metric for optimizing the protocol and to estimate the accuracy of the Nile red fluorescence, the geometric mean of the fluorescence distribution was compared to a chemical PHB measurement of the culture. As there is presently no validated method for measuring PHB levels at the individual cell level, population average measurements, such as the geometric mean of fluorescence and the whole-culture chemical PHB measurement, were required to assess the quantitative accuracy of the staining protocols. The correlation between fluorescence and PHB content was greatly improved over that of initial staining experiments (data not shown) due to the improved staining of PHB granules and reduction in nonspecific staining. The estimated error of prediction of PHB content from the geometric mean of fluorescence was ±1.2% PHB DCW and ±4.5% PHB DCW for Synechocystis
and E. coli
, respectively (95% confidence interval). From this, it can be inferred that the PHB levels on the single-cell level can be estimated accurately based on the fluorescence measurement.
These protocols will allow single-cell measurements of PHB levels in Synechocystis and E. coli to such a level of precision that mutants with incrementally increased PHB accumulation can be sorted from the library and characterized. Using FACS, 10 million cells can easily be assayed in less than 1 h. While there will be a loss due to nonviable cells in the E. coli system, this loss does not prohibit the assay from screening genome-scale libraries. As well, multiple cells of the same genotype will be present due to growth, increasing the likelihood of each library variant being screened.
Biological noise will most likely contribute false positives to the screen. Inherent in all single-cell measurements is the cell-to-cell variation even in a clonal population. This is evident in Fig. and . For the positive controls, a clonal population has a 10-fold difference in fluorescence within the population. This variation in PHB content will result in false positives being sorted as high-PHB clones, while their average PHB content may be less.
The application of sucrose shock to allow E. coli to take up Nile red is generalizable to other bacteria and other small-molecule dyes which do not permeate the membrane. By using such permeabilization methods to allow impermeant fluorescent dyes to enter the cytoplasm, the number of phenotypes that can be screened in a high-throughput fashion can be significantly increased. This will enable new fluorescence-based combinatorial screens for other phenotypes where high-throughput screens do not currently exist.