Team:Edinburgh/Directed Evolution
Directed evolution method development
The University of Edinburgh’s 2021 iGEM team also joined the International Directed Evolution competition (iDEC)
The iDEC competition, held for the first time in Edinburgh to commemorate the journey of the University of Edinburgh Alumni Charles Darwin, aims to encourage young students all around the world to harness the power of evolution through protein engineering techniques.
Danghatai (Baifern) Wongthanaroj, Charles Wackwitz, Davide Annese and Finn Slattery formed the iDEC team since each of them worked with directed evolution methods. For this competition they focused on two directed evolution methods, SeSaM (Sequence Saturation Mutagenesis) [1,2] and GeneORator [3]. However, the most important results achieved wasan alternative approach to SeSaM, leading them to win the “Special library construction” prize.
They managed to create an alternative procedure for the first step of the SeSaM method by substituting the dNTPαS + Iodine incubation step with sonication. This modification makes the sesam method faster, easier and cheaper, increasing its accessibility and applicability. They showed that the technique worked by sequencing 23 clones. This showcased different types of mutations among which an enhanced rate of transversions and 2 clones presenting consecutive mutations. Consecutive mutations are highly unlikely to happen with other directed evolution methods such as error-prone PCR, and so can likely be attributed to the SeSaM method. As a result, the University of Edinburgh’s 2021 IDEC team managed to obtain the silver medal in the final competition and won the library generation award.
Below is a brief description of the main results achieved. More information about the competition and the project can be found on the team’s iDEC wiki: https://idec2021.github.io/Edinburgh/
What is the Directed Evolution of proteins?
The directed evolution of proteins comprises a collection of molecular biology techniques that aims to improve a particular feature of a protein. This can generate a more robust, stable or catalytically active variant in comparison to its original wildtype. This is typically acquired through repeated cycles of mutagenesis, in which variants are screened to identify mutants with improved desirable features. In laboratory settings, directed evolution techniques can create an artificial human-controlled evolutionary pressure, and increase the mutation rate by isolating the gene from its original host, and creating a large library with various mutations. In this way we can simulate thousands or even hundreds of thousands of generations in a short period of time. The best mutants are selected for purpose while generating useful data about the structure and function of the protein itself.
Objective of the Project
To improve upon the mutational spectrum of error-prone polymerase chain reaction (epPCR) in generating libraries through the optimisation of the sequence saturation mutagenesis (SeSaM) protocol to induce mutations uncommon to Taq polymerase. Additionally, generate a semi-rationally designed mutant library for the eventual identification of positive variants capable of degrading recalcitrant polymers related to industrially relevant waste streams.
Main Results achieved
- Sonication as a novel fragmentation method
The gene of interest is fragmented in the SeSaM protocol in order to allow terminal transferase to elongate with a universal base on the 3' end of the forward fragment to induce mutagenesis. The original SeSaM protocol utilises dNTPαS incorporated into the gene of interest for subsequent random shearing across the full gene-length. However, owing to the high cost of these modified bases, an alternative DNA shearing method was performed by sonication to explore more accessible alternatives. Various rounds of sonication were performed and are depicted in Figure 1.
Figure 1 Fragmentation of DNA by Number of Sonication Cycles. 2% agarose gel showing the fragmented DNA after sonication. (1) 100 bp Promega DNA ladder, (2-10) 0 - 8 cycles of sonication: pulse on 15 sec, pulse off 60 sec, 20% amplitude.
Increasing the rounds of sonication decreases the concentration of the full-length gene and increases the size of the smear. In this case, a long smear with minimal concentration of the full-length gene is desirable to increase the mutational spectrum of the method. Eventually, The optimised protocol was followed with the sonicated fragments and was successfully used to generate a full-length gene product (Figure 2).
Figure 2 Final PCR product obtained from the optimised SeSaM. The Cex-alphaS full-length and Cex-sonicated full-length were loaded on gel in the equivalent amount as in the PCR mixture. pJUMP19_Cex was amplified as a positive control and both Cex-alphaS and Cex-sonicated were set in a PCR with three different concentrations of template: 1 ng, 30 ng, and 60 ng. The sample fragmented by sonication was successfully produced, however the sample fragmented by dATPαS always appeared as a smear for an unknown reason.
- Mutant Library Sequencing
The resulting product was successfully assembled via Golden Gate assembly and cloned into E. coli DH5α. Colonies with successful colony PCR results were miniprepped and sent for sequencing for the confirmation of mutations with the gene (Figure 3). The majority of the mutations are common substitution (in blue/light blue) which can be attributed to the three rounds of PCR steps in which a normal Taq polymerase was used. In fact, this polymerase is known to have natural flaws with high biases versus transition mutations. [4] However, the sequencing revealed that 2 of our clones had consecutive mutations, which are very unlikely to be related to polymerase mistakes, and the total transversions accounted for 20.5.
Figure 3 Mutational Spectrum of the Optimised SeSaM Method with Novel Sonication Shearing. Number and types of mutation found per clone sequenced (B) Total types of mutations found from sequencing of mutants.
- 1. Tuck Seng Wong, Kang Lan Tee, Berhard Hauer, Ulrich Schwaneberg, Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution, Nucleic Acids Research, Volume 32, Issue 3, 1 February 2004, Page e26, doi:10.1093/nar/gnh028
- 2. Wong TS, Roccatano D, Loakes D, et al. Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR. Biotechnol J. 2008;3(1):74-82. doi:10.1002/biot.200700193
- 3. Currin A, Kwok J, Sadler JC, et al. GeneORator: An Effective Strategy for Navigating Protein Sequence Space More Efficiently through Boolean OR-Type DNA Libraries. ACS Synth Biol. 2019;8(6):1371-1378. doi:10.1021/acssynbio.9b00063
- 4. Keohavong P, Thilly WG. Fidelity of DNA polymerases in DNA amplification. Proc Natl Acad Sci U S A. 1989;86(23):9253-9257 doi:10.1073/pnas.86.23.9253