Optimization of cell density for induction

We introduced another factor – cell density when seeking for a local optimum of induction time when performing induction in SG-SCAA at 30 °C to broaden the applicability of the optimized condition. Since most of the time yeast is inoculated from plates directly into culture tubes, it is impossible to check the starting yeast age. Indeed,

a little more HLA-DR1 molecules were detected when inoculating at a higher density (OD600~0.4) than lower one (OD600~0.05) (Figure 3-2D), partially reflecting that starting

with fresh cells tends to result better than older ones for a certain growth length, because old ones normally contain more dead cells which won’t divide. This postulation was also confirmed by the experimental result being talked about in Chapter IV (Figure 4-3). Bearing this in mind, we tried to inoculate yeast as fresh as possible henceforward. However, if cells are too old, several passages using fresh medium will always dilute out older ones, so it is not necessary to control the starting cell density for inoculation.

Densities of cells collected and initiated for induction are parameters that could affect the final HLA-DR1 surface display level. Yeast collected from SD-SCAA culture at both an OD600 of 3.0 and 6.0 exhibit optimal DR signals after being induced for 16 h

regardless of what OD600 used for initiating induction (Figure 3-3A and B), suggesting

that an optimal induction time could be somewhere closed to 16 h. A direct comparison of these two local optimums (Figure 3-3C) further confirms that cells collected at a smaller OD600~3.0 and induced in SG-SCAA at a smaller OD600~0.5 will display more

detectable HLA-DR1 than those with bigger OD600. Additionally, serving as a parallel

control, results of yeast induced in YPG at different initial densities (Figure 3-3D) confirmed that collecting cells at a relatively earlier growth stage helped to display more HLA-DR1. This makes us to speculate that cells should be collected and induced within the exponential phase of growth or before stationary phase, after which the changes in cell wall structure could possibly influence surface display and detection of target proteins.

Figure 3-3 Effect of densities of cells collected and initiated for induction on HLA-DR1 surface display level. Co-displaying yeast grown up in SD-SCAA were collected at either A. OD600 ~ 3.0 (open

symbols) or B. OD600 ~ 6.0 (filled symbols), spun down and resuspended in SG-SCAA to various densities

at an OD600 of 0.5, 1.0 or 2.0 as indicated, and then induced at 30°C for various time lengths. C. Further

comparison of the DR signal from yeast collected at the two different OD600 and induced with a starting

OD600 of 0.5. D. Cells collected at different OD600 after enrichment were also resuspended in different

batches of YPG media to an OD600 of 1.0 and induced at 20 °C for 20 h. After induction, all samples in

were collected and labeled at the same time for flow cytomeric analysis. cMFI(DR) were calculated as

described previously for yeast prepared at different conditions.

Before performing more experiments searching for optimal densities of cells collected and initiated for induction, we defined the growth pattern for co-displaying yeast strain in SD-SCAA medium. 2 ml of SD-SCAA fresh media in glass culture tubes were inoculated by two-day-old cell cultures (used to be kept at 4 °C) to an OD600 of

points plotted in log scale against growth time (Figure 3-4) reveals a curve for yeast growing in SD-SCAA medium. The curve is actually linear before 20 h (OD600~4.0),

reflecting an exponential growth phase, and then starts to tilt down a little bit, indicating the beginning of stationary phase or a deceleration stage for entering stationary, where apoptosis of older cells in the culture tube emerges. Furthermore, the doubling time for yeast cells growing in SD-SCAA can be determined using this curve, which is around 3.5 hours. It is clear that OD600~3.0 (~19 h post-inoculation) is within the exponential

phase whereas OD600~6.0 (~24 h post-inoculation) is towards the end of this partial

growth curve closed to the stationary phase, confirming our assumption that cells collected in exponential growth stage tended to display more HLA-DR1 than those collected outside.

Figure 3-4 Cell growth curve for co-displaying yeast cultured in SD-SCAA.

To further evaluate the effect of growth stage and starting cell density on induction of proteins expression and association, more OD600 were examined (Figure 3-5). It is

amount of detectable HLA-DR1 after proper induction, while a relative higher cMFI is observed at OD600~5.2 (Figure 3-5A). The suggested range of OD600 is about 2.0 to 6.0,

which is a little broader than that obtained from previous results. Meanwhile, the checking for different initial cell densities of induction culture shows an optimum around OD600~0.5 (Figure 3-5B), which is consistent with previous observation (Figure

3-3A).

Figure 3-5 Further evaluation for the effect of cell age and initial cell density. A. Cells were grown up to various ages (OD600), and then switched to SG-SCAA to an OD600 of 0.75. B. Cells were collected at an

OD600 of 3.7 and resuspended in SG-SCAA to various densities (OD600). In both experiments,

immunofluorescent labeling was performed using biotinylated LB3.1 and streptavidin-PE-Cy5. cMFI(DR)

were calculated and plotted against OD600 measurements representing densities of cells collected or

initiated for induction.

In sum, one local optimum condition for culturing co-displaying yeast would be: inoculate a 2 ml SD-SCAA medium by a single colony (or an aliquot from a few days old culture) in a culture tube and grow cells in 225 rpm shaker at 30 °C for 16~20 h until the density reach an OD600 of 2.5 to 5.0, then switch cells into 2 ml SG-SCAA to an