Modern methods for laboratory diversification of biomolecules

Bratulić, Siniša; Badran, Ahmed H.

doi:10.1016/j.cbpa.2017.10.010

Cited by 16 publications

(11 citation statements)

References 80 publications

(111 reference statements)

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“…In vivo mutagenesis methods require minimal intervention because selection and mutagenesis are coupled in the same system. Most in vivo mutagenesis methods suffer from low mutation rates and increased off-target mutagenesis 31 . Therefore, there is a need for an in vivo mutagenesis systems which can diversify a chosen gene of interest with a high frequency.…”

Section: Discussionmentioning

confidence: 99%

Transient AID expression for in situ mutagenesis with improved cellular fitness

Al-Qaisi

Roffler

2018

Sci Rep

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Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3–5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.

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Section: Discussionmentioning

confidence: 99%

Transient AID expression for in situ mutagenesis with improved cellular fitness

Al-Qaisi

Roffler

2018

Sci Rep

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“…Genetic diversification drives directed evolution, and success of the campaign heavily depends on the nature, size, and quality of the genetic library ( Bratulic and Badran, 2017 ). For single genes or shorter DNA fragments, the commonly used approaches of genetic diversification can be used ( Gillam et al, 2014 ).…”

Section: Challenges and Possible Solutionsmentioning

confidence: 99%

Roadmap to Building a Cell: An Evolutionary Approach

Abil

Danelon

2020

Front. Bioeng. Biotechnol.

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Laboratory synthesis of an elementary biological cell from isolated components may aid in understanding of the fundamental principles of life and will provide a platform for a range of bioengineering and medical applications. In essence, building a cell consists in the integration of cellular modules into system's level functionalities satisfying a definition of life. To achieve this goal, we propose in this perspective to undertake a semirational, system's level evolutionary approach. The strategy would require iterative cycles of genetic integration of functional modules, diversification of hereditary information, compartmentalized gene expression, selection/screening, and possibly, assistance from open-ended evolution. We explore the underlying challenges to each of these steps and discuss possible solutions toward the bottom-up construction of an artificial living cell.

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“…This technique has been applied to engineer RNA polymerases [ 38 ], aminoacyl-tRNA synthetases [ 39 ], and even toxins [ 40 ]. For more details, we refer to recent reviews [ 41 , 42 ].…”

Section: Creating Diversity: From Gene Level To Population Levelmentioning

confidence: 99%

Selecting the Best: Evolutionary Engineering of Chemical Production in Microbes

Shepelin

Hansen

Lennen

et al. 2018

Genes

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Microbial cell factories have proven to be an economical means of production for many bulk, specialty, and fine chemical products. However, we still lack both a holistic understanding of organism physiology and the ability to predictively tune enzyme activities in vivo, thus slowing down rational engineering of industrially relevant strains. An alternative concept to rational engineering is to use evolution as the driving force to select for desired changes, an approach often described as evolutionary engineering. In evolutionary engineering, in vivo selections for a desired phenotype are combined with either generation of spontaneous mutations or some form of targeted or random mutagenesis. Evolutionary engineering has been used to successfully engineer easily selectable phenotypes, such as utilization of a suboptimal nutrient source or tolerance to inhibitory substrates or products. In this review, we focus primarily on a more challenging problem—the use of evolutionary engineering for improving the production of chemicals in microbes directly. We describe recent developments in evolutionary engineering strategies, in general, and discuss, in detail, case studies where production of a chemical has been successfully achieved through evolutionary engineering by coupling production to cellular growth.

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Modern methods for laboratory diversification of biomolecules

Cited by 16 publications

References 80 publications

Transient AID expression for in situ mutagenesis with improved cellular fitness

Transient AID expression for in situ mutagenesis with improved cellular fitness

Roadmap to Building a Cell: An Evolutionary Approach

Selecting the Best: Evolutionary Engineering of Chemical Production in Microbes

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