Team:Edinburgh OG/Enzymes
Dye-Degrading Enzymes
Laccase enzymes are ‘green catalysts’ that are able to degrade a wide range of compounds, including azo dyes. Laccases are common in nature and have been found in bacteria, fungi, plants and insects. The properties of laccases vary depending on their host organism (Sharma et al., 2007). Fungal laccases have been a focus for recent bioremediation strategies due to their high catalytic activity. However, they lose their catalytic activity in extreme conditions. Additionally, through speaking with researchers in the Horsfall laboratory we found that fungal laccases are extremely difficult to express heterologously in bacterial chassis. Literature showed that bacterial laccases exhibit greater stability in extreme conditions and thus we decided to use the bacterial laccase Bpul from Bacillus pumilus. However, these enzymes have a lower redox potential than fungal laccases and degradation of dyes using bacterial laccases currently happens at an unsatisfactory rate. Therefore, we decided to take in vitro and in vivo directed evolution approaches to create a thermo- and pH-stable laccase with high catalytic activity. |
In Vitro
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Directed evolution is a method designed to mimic natural selection, resulting in a powerful tool for improving enzymes in the laboratory. Directed evolution consists in target a gene to iterative rounds of mutagenesis, selection and amplification. To prepare our gene for directed evolution we joined Bpul (K863001) with the leader sequence PelB (J32015) using Gibson Assembly. The PelB leader sequence directs the protein to the bacterial periplasm, where it will be released into the supernatant. This would provide us with a WT Bpul laccase that will be exported out of the cell, and a template for mutant library generation. In vitro mutagenesis was performed using error prone PCR. The theory behind error prone PCR is that an increase in Mg2+ and the addition of Mn2+ causes a reduction in the base pairing fidelity of Taq Polymerase. An advantage of error prone PCR is that error rates can be highly controlled by adjusting Mg2+ and Mn2+ ions for the protein of interest that is being mutated. The mutant library was then inserted into a pET28 expression vector using Golden Gate assembly before being transformed into BL21 DE3 chemically competent Escherichia coli via heat shock. To determine if mutagenesis produced functional laccases a plate-based dye degradation assay. Improvement in laccase activity was determined by ABTS assay and those mutants with increased activity were selected and remutated to seek further improvement. ExperimentsBpul laccase and the PelB leader sequence were taken from the iGEM kit (part registry: K863001 and J32015). They were resuspended in 10 μL of nuclease free water and incubated at room temperature for 1 h. Resuspended DNA was directly amplified with Q5 PCR (New England BioLabs) (1X Q5 Rection Buffer, 200 μM dNTP mix, 0.5 μM PelB Forward and PelB Reverse or, Bpul Forward and Bpul Reverse, 10 μL of resuspended DNA, and 0.5 μL of Q5 Polymerase) in a 50 μL reaction volume. The PCR cycle consisted of an initial denaturation step at 98℃ for 30 s. Followed by 35 cycles of 98℃ for 10 s, 72℃ for 30 s, and 72℃ for 30 s. This was followed by a final elongation step at 72℃ for 2 min. Parts were assembled via Gibson Assembly following the Gibson Master Mix protocol (New England Biolabs). The reaction mixture consisted of 0.5 pm of Bpul and PelB fragments in a 1:1 ratio, 1X Gibson Assembly Master Mix, and deionised water up until a final volume of 20 μL. Samples were incubated at 50℃ for 15 min. Assembly of parts was verified by agarose gel electrophoresis (2 %) stained with SYBER Safe gel stain (Thermo Fisher) and imaged with a BioDoc-It Imaging Systems benchtop UV transilluminator (UVP). Libraries of laccase mutants were generated using error prone Taq PCR (New England BioLabs). In a 50 μL reaction volume; 1X NEB Taq Reaction Buffer, 5 μL of dNTP mix (3.5 mM dATP, 4 mM dCTP, 6 mM dGTP, and 13.5 mM dTTP), 2.95 mM MgCl2 , 0.1 mM MnCl2, 0.2 μM forward and reverse primer, 0.5 μL of NEB Taq Polymerase, and 50 ng of DNA template was added. The PCR cycle consisted of an initial denaturation step of 95℃ for 1 min. Followed by 25 cycles of denaturation at 95℃ for 30 s, annealing at 59℃ for 45 s, and elongation for 90 s at 68℃. This was followed by a final elongation step at 68℃ for 5 min. The mutant libraries of assembled PelB and Bpul were then cloned into pET28a-GG-RFP-CD via Golden Gate cloning. BsaI restriction sites were added to the Gibson Assembly product via Q5 PCR. The reaction cycle and reaction mixture were the same as described above, with Bpul GG Forward and Bpul GG Reverse primers. Golden Gate cloning was performed in 20 μL reaction volumes consisting of; Bpul insert and pET28a-GG-RFP-CD vector in a 3:1 ratio, 1X T4 ligase buffer, 10 U BsaI-HFv2 (New England Biolabs), 400 U T4 DNA Ligase (New England Biolabs). The Golden Gate reaction cycle consisted of, 15 cycles of 37℃ for 1.5 min and 16℃ for 3 min. Followed by 5 min at 50℃, and 80℃ for 10 min. The Golden Gate reaction was directly used for bacterial transformation.
Figure 1. Schematic showing the stages of in vitro directed evolution via error-prone PCR mutagenesis. PCR is followed by cloning, screening, and then more PCR to introduce more mutation. Results
A) Figure 2. Results of in vitro directed evolution of laccase. A)For in vitro directed evolution 94 mutants were screened via plate-based dye degradation assays. From these assays 23 active clones were observed. B) These active laccase enzymes were assayed for enzymatic activity using ABTS assay. This assay showed four mutants with improved activity (one with a significant increase). C)This mutant was sequenced and showed eight-point mutations when compared to WT Bpul. |
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In Vivo
Directed evolution is a method designed to mimic natural selection, resulting in a powerful tool for improving enzymes in the laboratory. Directed evolution consists in target a gene to iterative rounds of mutagenesis, selection and amplification. To prepare our gene for directed evolution we joined Bpul (K863001) with the leader sequence PelB (J32015) using Gibson Assembly. The PelB leader sequence directs the protein to the bacterial periplasm, where it will be released into the supernatant. This would provide us with a WT Bpul laccase that will be exported out of the cell, and a template for mutant library generation.
Figure 1. Overview of the temperature sensitive switch capable of the JS200 mutator strain with the temperature sensitive allele of PolI (polA12). Increasing the temperature to 37 oC reduces the activity of polA12 and epPolI becomes the predominant activity, resulting in mutations. ExperimentsMutant libraries of laccase were also generated in vivo using JS200 E. coli. This is an E. coli with an error prone polymerase I enzyme which results in faulty replication causing random mutations. As polymerase I solely replicates plasmids with a ColE1 origin, this means this method of mutagenesis is targeted and does not have possess the downsides of other mutators strains such as XL1-Red. Additionally as the JS200 strain possess a recA mutation it means its native polymerase I is temperature sensitive and so at 37 oC the error-prone polymerase I becomes the dominant activity, this allows for control of mutagenesis. For library generation Bpul and PelB were inserted into plasmids containing ColE1 origin and incubated at 37 C to facilitate mutagenesis. Mutants were plated and plasmids recovered before transformation into expression E. coli strains for activity assays.
Figure 2. Steps involved for generating mutant library using JS200 mutator strain 1) Transform target plasmid into wtPolI and epPolI plasmid containing JS200 cells 2) Incubate at 37 |
