Directed evolution
Directed evolution (DE) is a method used to produce enzymes for industrial or medical purposes.
The method is protein engineering which mimics natural selection.[1]
The basic idea is to put a gene through repeated rounds of mutation, to make a library of variants. Selection isolates genes with the desired function. They are a template for the next round.[2]
This can be done in vivo (in living cells of bacteria or yeast), or in vitro (free in solution or microdroplets).
Testing more mutants increases the chances of finding one with the desired properties.[3]
During in vivo evolution, each cell (usually bacteria or yeast) is transformed with a plasmid containing a different member of the variant library. Only the gene of interest differs between the cells, with all other genes being kept the same.
The cells express the protein either in their cytoplasm or surface where its function can be tested. This format has the advantage of selecting for properties in a cellular environment, which is useful when the evolved protein or RNA is to be used in living organisms.[4]
When done without cells, DE uses in vitro transcription translation to produce proteins or RNA free in solution or inside artificial microdroplets. This has the benefit of allowing more conditions (e.g. temperature, solvents). It can express proteins that would be toxic to cells. Furthermore, in vitro evolution experiments can generate far larger libraries (up to 1015) because the library DNA need not be inserted into cells. That often limits what can be done.
Ensuring heredity
change
Either way the gene which codes for the protein is isolated
When functional proteins have been isolated, it is necessary that their genes are too, therefore a genotype-phenotype link is required.[5]
This can be covalent, where the mRNA gene is linked to the protein at the end of translation by puromycin.[6]
Alternatively the protein and its gene can be kept together,[7] or in emulsion droplets.[8] The gene sequences isolated are then multiplied by PCR or by transformed host bacteria. Either the single best sequence, or a pool of sequences can be used as the template for the next round of mutagenesis. The repeated cycles of diversification-selection-amplification make enzyme variations adapted to the selection process.
Prize awarded
changeUS engineer Frances Arnold has won the Millennium Technology Prize for pioneering directed evolution.[9]
References
change- ↑ Stephen Lutz 2010. Beyond directed evolution - semi-rational protein engineering and design. Curr Opin Biotechnol. 21(6): 734–743.
- ↑ Voigt C.A; Kauffman S. & Wang Z.G. 2000 (2001). "Rational evolutionary design: the theory of in vitro protein evolution". Advances in Protein Chemistry. 55: 79–160. doi:10.1016/s0065-3233(01)55003-2. ISBN 9780120342556. PMID 11050933.
{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: numeric names: authors list (link) - ↑ Dalby P.A. 2011 (2011). "Strategy and success for the directed evolution of enzymes". Current Opinion in Structural Biology. 21 (4): 473–80. doi:10.1016/j.sbi.2011.05.003. PMID 21684150.
{{cite journal}}: CS1 maint: numeric names: authors list (link) - ↑ Badran A.H. & Liu D.R. 2014 (2015). "In vivo continuous directed evolution". Current Opinion in Chemical Biology. 24C: 1–10. doi:10.1016/j.cbpa.2014.09.040. PMC 4308500. PMID 25461718.
{{cite journal}}: CS1 maint: numeric names: authors list (link) - ↑ Leemhuis H. et al 2005 (2005). "New genotype-phenotype linkages for directed evolution of functional proteins". Current Opinion in Structural Biology. 15 (4): 472–8. doi:10.1016/j.sbi.2005.07.006. PMID 16043338.
{{cite journal}}: CS1 maint: numeric names: authors list (link) - ↑ Lipovsek D. & Plückthun A. 2004 (2004). "In-vitro protein evolution by ribosome display and mRNA display". Journal of Immunological Methods. 290 (1–2): 51–67. doi:10.1016/j.jim.2004.04.008. PMID 15261571.
{{cite journal}}: CS1 maint: numeric names: authors list (link) - ↑ Nguyen A.W. & Daugherty P.S. 2005 (2005). "Evolutionary optimization of fluorescent proteins for intracellular FRET". Nature Biotechnology. 23 (3): 355–60. doi:10.1038/nbt1066. PMID 15696158. S2CID 24202205.
{{cite journal}}: CS1 maint: numeric names: authors list (link) - ↑ Schaerli Y. & Hollfelder F. 2009 (2009). "The potential of microfluidic water-in-oil droplets in experimental biology". Molecular BioSystems. 5 (12): 1392–404. doi:10.1039/b907578j. PMID 20023716.
{{cite journal}}: CS1 maint: numeric names: authors list (link) - ↑ Webb, Jonathan 2016. Evolutionary engineer Frances Arnold wins €1m tech prize. BBC News Science & Environment. [1]