A population of gus-wt, prepared by SSM, was ligated into pJWL1030 via NdeI and XbaI restriction sites. The variants of gus-wt were created and resulting in pJWL1030-guswt* constructs. In contrast, the random mutagenesis libraries of gus-wt and gus-tr3337 were digested at XbaI and XhoI restriction sites and ligated into the pET28a(+) expression vector, resulting in pET28a-guswt* and pET28a-gustr3337*, respectively. The ligation reaction products were isolated with a Promega® PCR purification kit and eluted in 30 μL of water. 5 μL of the purified ligation products was transformed into 50 μL of E. coli strain GMS407 and recovered with 1 mL yeast extract nutrient broth (YENB) (Appendix B) at 37 ˚C. 50 μL of the culture was plated out on Luria-Bertani-Kanamycin (LBK) agar (Appendix B) and incubated overnight at 37 ˚C to estimate the cell density. After determining the cell density, the transformants were diluted such that 50 μL of culture that contained about 300 viable cells. Library culture aliquots of 50 μL were applied to each LB-Kanamycin agar and spread out with sterile ball bearings (5 mm diameter, steel) to provide an even distribution of colonies. 10 colonies were sequenced to examine the randomness of the point mutations; that is the average number of mutations.
2.3.1.2.1. In vitro screening of site-directed libraries with β-glycoside substrates
In each SSM library, 96 single colonies were picked manually from the LBK agar plates and grown individually in 96-well round bottom culture plates with 200 μL of LBK per well. The plates were incubated at 37 ˚C overnight. 120 μL of culture from each well was transferred to a new sterile 96-well microplates and 20 μL of 7x BugBuster was added to each well to lyse the
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cells. The proteins were assayed with 200 μM para-nitrophenyl-β-D-glucuronide (pNP- glucuronide), 3 mM pNP-glucopyranoside, 3 mM pNP-galactopyranoside, 3 mM pNP- mannopyranoside and 3 mM pNP-xylopyranoside in 50 mM phosphate buffer, pH 7.4. The release of pNP was monitored at 405 nm (A405) on a Labsystems Multiskan Ascent 96-well
microplate ultraviolet/visible spectrometer. The activities were compared to those of wild- type. Clones with enhanced, moderate and decreased activities toward five substrates were selected. Plasmids were isolated with miniprep kit and sequenced.
2.3.1.2.2. Colony screening
E. coli expressing β-GUS can moderately cleave externally supplied 5-bromo-4-chloro-3-indolyl-
β-D-glucoside (X-glucoside) to produce glucose and an intense blue precipitate of chloro- bromoindigo. Another artificial chromogenic substrate, pNP-glucoside, can also be moderately hydrolysed by β-GUS resulting in glucose and the yellow stained pNP.
This formed the primary screening and secondary phases. The primary screening can be done on solid medium that contains X-glucoside whereas the liquid medium base for secondary screening contained pNP-glucoside. The primary screening narrows hundreds of thousands of β-GUS versions down to less than three thousand.
2.3.1.2.2.1. Primary screening of colonies
The first two generation examined colonies via agar plate screening while generations 3, 4 and 5 examined colonies via top agar screening. An overview of this scheme is presented in the Figure 2.2.
Figure 2.2 Screening strategies
The two screening strategies used in directed evolution of GUS-WT and GUS-TR3337 are presented in a schematic form with agar plates, top agars plates and 96-well plates are illustrated at the top, bottom and right, respectively. Agar plate screening is described in Section 2.3.1.2.2.1.1., top agar screening is described in Section 2.3.1.2.2.1.2. and culture screening is described in Section 2.3.1.2.2.2.
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2.3.1.2.2.1.1. Agar plate screening
50 μL of library culture aliquots were applied to each agar plate. The agar plates were prepared with 10 mL of LBK agar supplemented with 0.15 mM IPTG and 0.1 mM X-glucoside. After overnight incubation at 37 ˚C, thirty most blue colonies on each plate were picked and inoculated in individual 200 μL aliquots of sterile LBK into a 96-well culture plate.
Each screening batch, typically a 100 agar plates, resulted in about 3000 inoculants with additional inoculants as controls. The negative control was E. coli GMS407 transformed with empty pET28a(+) and the positive control was the same strain expressing GUS-WT, GUS- TR3337 or the best variant from each generation.
2.3.1.2.2.1.2. Top agar screening
15 mL of molten glucosidase activity indicator agar (Appendix A) was carefully poured onto each agar plate, covering all the colonies. Colonies expressing active β-GUS would start to turn darker blue after 10 min with the top agar. The two most blue and distinct colonies on each plate were identified within 30 min. Each of the identified colonies was used to inoculate individual 200 μL aliquots of sterile LBK (Appendix B).
2.3.1.2.2.2. Secondary screening of β-GUS activity
After the 96-well culture plates were incubated overnight, 120 μL of each culture was transferred to a 96-well plate. The cells of each sample were lysed by adding 20 μL of 7x BugBuster and incubating for 30 min at room temperature. Addition of 100 μL of assay buffer produced a 220 μL reaction with: 50 mM phosphate buffer at pH 7.4, 800 μM of pNP- glucoside. The reactions were performed at room temperature (25 ˚C) and the hydrolysis of pNP-glucoside was measured by monitoring the rate of the release of pNP at A405 over 20 min.