Developing Non-selective Growth Conditions

With optimized and more stringent selection conditions established, we next turned to optimizing non-selective conditions for post-transformation rescue and out-growth steps. To prevent large fractions of the library population from dropping out before the first timepoint on the turbidostat, we sought to identify supplements to M9 minimal medium that would keep the growth rates of highly-fit and unfit mutants within a factor of 2. With this criterion and a WT growth rate in the range of 0.3-0.5 hr≠1, the relative frequency of alleles would not change by more than 10- to

20-fold between transformation and the first timepoint on the turbidostat. Additionally, because post-transformation rescue in SOB medium is necessary for sufficient transformation efficiency, we aimed for non-selective out-growth conditions in M9 medium to allow the selection strain to adapt to glucose as a carbon source. We first attempted to use a previously reported supplement mix ("folA mix") of 100 µg/mL (1 mM) adenine, 250 µg/mL (1 mM) thymidine, 1 µg/mL (2

µM) pantothenate, 38 µg/mL (500 µM) glycine, and µg/mL (250 µM) methionine[33]. From

plate reader growth experiments with these supplements, we observed that the unfit F31Y/L54I mutant (see Table 3.1, page 65) had a measureable growth rate that was within 2-fold of that for WT DHFR (Figure 3.15, page 75), but the selection strain displayed a significant lag phase both for moderately active DHFR mutants (Figure 3.16, page 75) and for a monoculture of WT DHFR in the turbidostat (Figure 3.17, page 76). From these results, we concluded that the individual components of this published supplement should be optimized individually.

We examined the supplement components and their concentration, and hypothesized that the high concentration of nucleotides could cause toxicity by perturbing the nucleotide pools[? ]. We therefore decreased the nucleotide concentrations, resulting in a supplement mix of 20

Figure 3.15: Selection strain growth rates in M9 minimal medium without and with the previously pubmished "folA" supplement mix are shown as box plots. ER2566 folA/ thyA is complemented with one of 4 DHFR variants (labels bottom, Table 3.1, page 65) on the SMT205 optimized selection plasmid (Table 5.2, page 174) and grown in M9 minimal medium. For each DHFR variant, box plots represent 8 individual experiments on the plate reader (see Methods, Chapter 3.5.0.4, page 110). An orange line marks the median. Box edges mark the first and third quartiles. Whiskers mark the maximum and minimum.

Figure 3.16: Selection strain growth curves in M9 minimal medium without and with the previously published "folA" supplement mix. ER2566 folA/ thyA is complemented with one of 4 DHFR variants (labels bottom,

Table 3.1, page 65) on the SMT205 optimized selection plasmid (Table 5.2, page 174) and grown in M9 minimal

medium. For each DHFR variant, growth curves are shown for 8 individual experiments on the plate reader (see

Figure 3.17: Growth rates in the turbidostat after overnight growth in M9 minimal medium supplemented with the "folA mix". ER2566 folA/ thyA was transformed with WT DHFR in the SMT205 selection plasmid (Table

5.2, page 174) and grown in M9 minimal medium under turbidostat clamp after overnight growth and 4 hours

out-growth in M9 media with 100 µg/mL adenine, 250 µg/mL thymidine, 1 µg/mL pantothenate, 38 µg/mL glycine, and 38 µg/mL methionine. Points represent the growth rate calculated as the linear regression slope of Log2(ABS600). Error bars represent the standard error from linear regression, and the size of the marker is scale

by the Pearson R2 _value.

µg/mL (500 µM) glycine, and 38 µg/mL (250 µM) methionine. In plate reader growth

experiments (see Methods, Chapter 3.5.0.4, page 110) with this supplemented M9, we observed a measurable growth rate for the F31Y/L54I mutant that was not observed in the absence of thymidine or adenosine (Figure 3.18, page 77). Furthermore, the growth rate with F31Y/L54I was reproducibly 60% of the growth rate with WT when using the fully supplemented M9 medium. The doubling period with WT was 1.7 hours, and the doubling period with F31Y/L54I was 2.5 hours (Figure 3.19, page 77). During an estimated 16-hours of rescue and overnight out-growth, the selection strain complemented with WT DHFR would go through 9.4 generations versus 6.4 generations with F31Y/L54I. The difference in 3 generations would result in an 8-fold change in relative frequency in the library. Finally, a monoculture of the selection strain in the turbidostat after out-growth in supplemented M9 medium did not

Figure 3.18: Selection strain growth curves in supplemented M9 minimal medium without and with adenosine and thymidine drop-outs to the medium. ER2566 folA/ thyA is complemented with one of 4 DHFR variants (labels bottom, Table 3.1, page 65) on the SMT205 optimized selection plasmid (Table 5.2, page 174) and grown in M9 minimal medium with A) 20 µg/mL adenosine, 50 µg/mL thymidine, 1 µg/mL pantothenate, 38 µg/mL glycine, and 38 µg/mL methionine; B) the supplements from A) minus adenosine; or C) the supplements from A) minus thymidine. For each DHFR variant, growth curves are shown for 3 individual experiments on the plate reader (see Methods, Chapter 3.5.0.4, page 110).

Figure 3.19: Selection strain growth rates in M9 minimal medium. A) Growth rates for ER2566 folA/ thyA complemented with one of 4 DHFR variants (labels bottom, Table 3.1, page 65) on the SMT205 optimized selection plasmid (Table 5.2, page 174) and grown in M9 minimal medium with 20 µg/mL adenosine, 50 µg/mL thymidine, 1 µg/mL pantothenate, 38 µg/mL glycine, and 38 µg/mL methionine. For each DHFR variant, box plots represent 3 individual experiments on the plate reader (see Methods, Chapter 3.5.0.4, page 110). An orange line marks the median. Box edges mark the first and third quartiles. Whiskers mark the maximum and minimum. B) Correlation between growth rates from A) and in vitro enzyme velocity at 10 µM DHF calculated from Michaelis-Menten kinetics (Table 3.1, page 65). Plot points are colored by mutant identity (legend, right).

Figure 3.20: Growth rates in the turbidostat after overnight growth in supplemented M9 minimal medium. ER2566 folA/ thyA was transformed with WT DHFR in the SMT205 selection plasmid (Table 5.2, page 174) and grown in M9 minimal medium under turbidostat clamp after overnight growth and 4 hours out-growth in M9 media with 20 µg/mL adenosine, 50 µg/mL thymidine, 1 µg/mL pantothenate, 38 µg/mL glycine, and 38 µg/mL methionine. Points represent the growth rate calculated as the linear regression slope of Log2(ABS600). Error

bars represent the standard error from linear regression, and the size of the marker is scale by the Pearson R2

value.