Selection platforms for directed evolution in synthetic biology

Tizei, Pedro A. G.; Csibra, Eszter; Torres, Leticia L.; Pinheiro, Vitor B.

doi:10.1042/bst20160076

Cited by 78 publications

(73 citation statements)

References 123 publications

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“…This is commonly done in a time-consuming trial-and-error approach. To make this optimization process more efficient, synthetic biologists increasingly rely on directed evolution [5][6][7][8][9][10][11] . Here, genetic diversity is introduced to generate a library containing many variants of a synthetic construct or gene.…”

Section: Introductionmentioning

confidence: 99%

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology

2017

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Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

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Section: Introductionmentioning

confidence: 99%

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology

2017

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“…We acknowledge the limitations of using just one RNA and protein pair for analysis, the mutagenesis could be repeated with different fluorescent RNAs and phylogenetically distinct fluorescent proteins or other comparable functions such as self-splicing introns and inteins. Furthermore, simulated evolution experiments such as and directed evolution could be performed to identify differences between protein and RNA evolvability and robustness (56) . Alternatively, experimental evolution datasets could be mined for appreciable differences between protein and RNA evolution (57) .…”

Section: Discussionmentioning

confidence: 99%

The genetic robustness of RNA and protein from evolutionary, structural and functional perspectives

Sibaeva

McGimpsey

et al. 2018

Preprint

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The reactions of functional molecules like proteins and RNAs to mutation affect both host cell viability and biomolecular evolution. These molecules are considered robust if function is maintained despite mutations. Proteins and RNAs have different structural and functional characteristics that affect their robustness, and to date, comparisons between them have been theoretical. In this work, we test the relative mutational robustness of RNA and protein pairs using three approaches: evolutionary, structural, and functional. We compare the nucleotide diversities of functional RNAs with those of matched proteins. Across different levels of conservation, we found the nucleotide-level variations between the biomolecules largely overlapped, with proteins generally supporting more variation than matched RNAs. We then directly tested the robustness of the protein and RNA pairs with in vitro and in silico mutagenesis of their respective genes. The in silico experiments showed that proteins and RNAs reacted similarly to point mutations and insertions or deletions, yet proteins are slightly more robust on average than RNAs. In vitro , mutated fluorescent RNAs retained greater levels of function than the proteins. Overall this suggests that proteins and RNAs have remarkably similar degrees of robustness, with the average protein having moderately higher robustness than RNA as a group. Significance StatementThe ability of proteins and non-coding RNAs to maintain function despite mutations in their respective genes is known as mutational robustness. Robustness impacts how molecules maintain and change phenotypes, which has a bearing on the evolution and the origin of life as well as influencing modern biotechnology. Both protein and RNA have mechanisms that allow them to absorb DNA-level changes. Proteins have a redundant genetic code and non-coding RNAs can maintain structure and function through flexible base-pairing possibilities. The few theoretical treatments comparing protein and RNA robustness differ in their conclusions. In this experimental comparison of protein and RNA, we find that they have remarkably similar degrees of overall genetic robustness.

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“…Functional variants are identified by performing alternate cycles of positive selections in the presence of a ncAA and negative selections in the absence of a ncAA (77). Recent advances in directed evolution methods (114, 141) such as multiplex automated genome engineering (MAGE; 3, 132) and phage-assisted continuous evolution (29) may accelerate evolution. In addition to the traditional methods that use E. coli and yeast in vivo, positive in vitro selection systems were demonstrated as useful (28, 146), whereas negative selections are still limited to in vivo experiments (83).…”

Section: Expanding the Genetic Code With Orthogonal Translation Systemsmentioning

confidence: 99%

Rewriting the Genetic Code

Mukai

Lajoie

Englert

et al. 2017

Annu. Rev. Microbiol.

145

137

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The genetic code—the language used by cells to translate their genomes into proteins that perform many cellular functions—is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

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Selection platforms for directed evolution in synthetic biology

Cited by 78 publications

References 123 publications

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology

Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology

The genetic robustness of RNA and protein from evolutionary, structural and functional perspectives

Rewriting the Genetic Code

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