The gus-wt gene (GUS-WT) was manipulated using the construct created by cloning it into the pET28a(+) vector using the restriction sites NdeI and XbaI. Five methods were used to create variants of gus-wt and gus-tr3337 (designated gus-wt* and gus-tr3337*, respectively) and are outlined below.
2.3.1.1.1. Site-saturation mutagenesis (SSM)
β-GUS residues E413, N412, D163, R562, Y472, N566, T509, Y469, S557 and M447 were subjected to randomisation with SSM (Zheng et al. 2004). Degenerate primers containing the complete combination of 32 codons at the mutation points are designed to generate the SSM
25 libraries. Sets of offset NNK mutagenic primers (Table 2.1) were used to PCR amplify the entire gene and plasmid sequence. The product was digested with DpnI then purified and transformed into DH5α E. coli.
Table 2.1 Mutagenic NNK primers for SSM mutagenesis
Primer Sequence (5’ – 3’)
GUS E413 NNK F tat tgc caa cnn kcc gga tac ccg tc GUS N412 NNK F gag tat tgc cnn k ga acc gga tac cc GUS D163 NNK F agt taa aga ann kat gga agt aag ac GUS R562 NNK F aag gca tat tgn nkg ttg gcg gta ac GUS Y472 NNK F tta cgg atg gnn kgt cca aag cgg cg GUS N566 NNK F ctc gca agg cnn ktt gcg cgt tg GUS T509 NNK F gcg tgg atn nkt tag ccg ggc tgc a GUS Y469 NNK F gaa ccg tta tnn kgg atg gta tgt cca aag c GUS S557 NNK F ttt tgc gac cnn kca agg cat att gcg c GUS M447 NNK F gcg tca atg tan nkt tct gcg acg ct pJWL1030GUSHindIII R aag ctt ttg cca ttc tca ccg gat tc
SSM-PCR reactions were set up with 10 ng template pJWL1030-gus, 0.2 mM dNTP, 2.5U Pfu DNA polymerase, 1 μM forward and reverse primers, and 5 μL 10x Pfu reaction buffer and made up to 50 μL with deionised water. The SSM-PCR reaction mixtures were subjected to single 2-stage whole-plasmid PCR: 1 cycle at 95 ˚C for 3 min, followed by 5 cycles of 95 ˚C for 30 s, 55 ˚C for 1 min, 72 ˚C for 1 min/kb of mega-primer length, and 20 cycles of 95 ˚C for 30 s, 68 ˚C for 1 min/kb of template length, lastly, 1 cycle at 68 ˚C for 10 min.
2.3.1.1.2. Site-directed mutagenesis (SDM)
gus-tr3337, wt3p24e11-E504A, wt3p24e11-E504G, wt3p24e11-E504S, wt5p26a7-E504A, wt5p26a7-E504G, wt5p26a7-E504S, thermo4p11f2-E504A, thermo4p11f2-E504G and thermo4p11f2-E504G were constructed using SDM that was performed by the overlap extension PCR method with gus-wt as the DNA template and the primers listed in Table 2.2 (Xiong et al. 2004). The mutation site was introduced into the first PCR fragments, and the second fragment was amplified by annealing the overlapping ends of the first fragments. Final products were ligated into the pET28a(+) vector and sequenced. The strategy for the SDM was shown in Figure 2.1.
26
Table 2.2 Primers used for point mutations in site-directed mutagenesis Primer Sequence (5’ – 3’)
GUS Q493R F tgg cct ggc ggg aga aac tgc atc ag GUS Q493R R atg cag ttt ctc ccg cca ggc cag aag ttc GUS T509A F ccc ggc taa cgc atc cac gcc gta tt GUS T509A R ggc gtg gat gcg tta gcc ggg ctg GUS M532T F tgg ctg gat acg tat cac cgc gtc ttt g GUS M532T R gac gcg gtg ata cgt atc cag cca tgc ac GUS N550S F cag gta tgg agt ttc gcc gat ttt gc GUS N550S R aaa atc ggc gaa act cca tac ctg ttc ac GUS G559S F gac ctc gca aag cat att gcg cgt tgg GUS G559S R aac gcg caa tat gct ttg cga ggt cgc aa GUS N566S F gtt ggc ggt agc aag aaa ggg atc ttc GUS N566S R gat ccc ttt ctt gct acc gcc aac gcg pET28aGUS F aag aag gag ata tac cat ggg cag c pET28aGUS R tgg tgg tgc tcg agt cat tgt ttg cc
Figure 2.1 SDM from gus-wt and gus-tr3337 primers employed in this study Red: forward; blue: reverse.
2.3.1.1.3. Error-prone polymerase chain reaction (ePCR)
ePCR was based on the protocol developed by Leung et al. (1989), with reactions consisting of: 16 ng of the parent gene, 10 μM of forward and reverse primers, 0.2 mM of dNTPs, 5 mM MgCl2, different concentrations of Mn2+ ranging from 0.05 - 0,5 mM, 5 μL of 10x polymerase
buffer without Mg2+, 2.5 U Taq DNA polymerase and made up to 50 μL with deionised water. Thermocycling was performed with 30 cycles of 94 ˚C for 10 s, 50 ˚C for 10 s and 2 min at 72 ˚C. The ePCR products were purified with PCR purification kit and digested in one reaction with NdeI, XbaI and DpnI. The latter was required to selectively digest the parent genes since they were synthesised in vivo and have methylated DpnI restriction sites (Sambrook & Russell 2001). Finally, DNA with the correct size was isolated by gel purification with Promega® PCR purification kit and ligated into pET28a(+) with T4 ligase. The constructs
27 used in verifying the error-rate by DNA sequencing were purified from randomly selected GMS407 transformants.
2.3.1.1.4. Staggered extension process (StEP)
The StEP reaction consisted of 16 ng of template, 10 μM of each forward and reverse primer, 0.2 mM dNTP, 5 mM MgCl2 and 1 U of Phusion high fidelity polymerase. The cycling conditions
were 100 cycles of 94 °C for 10 s, 50 °C for 10 s and 72 ° for 2 s.
2.3.1.1.5. StEP-ePCR
Recombination of gene variants was simplified by the use of the StEP-ePCR. StEP-ePCR was set up in 50 μL with: 16 ng of parent genes in equal proportions, 10 μM of forward and reverse primers, 0.2 mM of dNTPs, 5 mM MgCl2, 1.5 mM MnCl2, 5 μL of 10x polymerase buffer without
Mg2+ and 2.5 U of Taq polymerase. SteP-ePCR thermocylcing required 30 cycles of: 94 ˚C for 10 s, 50 ˚C for 10 s and 72 ˚C for 10 s.