Library Generation by Gene Shuffling

Meyer, Adam; Ellefson, Jared W.; Ellington, Andrew D.

doi:10.1002/0471142727.mb1512s105

Cited by 20 publications

(23 citation statements)

References 14 publications

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“…Recloning allows the selection plasmid to be reset each round, thus preventing the accumulation of cheaters, and offers the opportunity to change the DNAP as needed. In some cases, in an effort to combine multiple beneficial mutations into a single variant, gene shuffling 54 was used between rounds (Methods).…”

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confidence: 99%

Marionette:E. colicontaining 12 highly-optimized small molecule sensors

Meyer

Segall-Shapiro

2018

Preprint

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2 Cellular processes are carried out by many interacting genes and their study and optimization requires 29 multiple levers by which they can be independently controlled. The most common method is via a 30 genetically-encoded sensor that responds to a small molecule (an "inducible system"). However, these 31 sensors are often suboptimal, exhibiting high background expression and low dynamic range. Further, 32 using multiple sensors in one cell is limited by cross-talk and the taxing of cellular resources. Here, we 33 have developed a directed evolution strategy to simultaneously select for less background, high 34 dynamic range, increased sensitivity, and low crosstalk. Libraries of the regulatory protein and output 35 promoter are built based on random and rationally-guided mutations. This is applied to generate a set 36 of 12 high-performance sensors, which exhibit >100-fold induction with low background and cross-37 reactivity. These are combined to build a single "sensor array" and inserted into the genomes of E. coli 38 MG1655 (wild-type), DH10B (cloning), and BL21 (protein expression). These "Marionette" strains allow 39for the independent control of gene expression using 2,4-diacetylphophloroglucinol (DAPG), cuminic 40 Advances in biology are often tied to new methods that use external stimuli to control the levels 45 of gene expression [1][2][3] . Pioneered in the early 1980s, so-called inducible systems were developed that allow 46 genes to be turned on by adding a small molecule inducer to the growth media 4 . These consist of a protein 47 transcription factor (e.g., LacI) whose binding to a DNA operator in a promoter is controlled by the inducer 48 (e.g., IPTG). Initially co-opted from natural regulatory networks, over the years many versions were 49 designed to improve performance. In the 1990s, additional systems were developed that responded to 50 other inducers, notably arabinose and aTc, which became common tools in the field. In 1997, Lutz and 51Bujard published a seminal paper that combined three (IPTG, arabinose, aTc) that could be easily 52 interchanged on a two-plasmid system 5 . Its organizational simplicity, compatibility, and quantified 53 response functions were revolutionary. Beyond providing a new tool to biologists to control multiple 54 genes with independent "strings," it facilitated researchers with quantitative backgrounds to enter 55 biology [6][7] . Armed with the new ability to control two genes with precision, physicists and engineers built 56 the first synthetic genetic circuits, performed single molecule experiments inside cells, deconstructed the 57 origins of noise in gene expression, determined how enzyme balancing impacts metabolic flux, elucidated 58 rules underlying the assembly of molecular machines, and built synthetic symbiotic microbial 59 communities, just to highlight a few [8][9][10][11][12][13][14][15][16][17][18][19] . 60. 20, 2018; 3 Sensor performance is quantified by its response function; in other words, how the concentration 61 of inducer changes th...

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Marionette:E. colicontaining 12 highly-optimized small molecule sensors

Meyer

Segall-Shapiro

2018

Preprint

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“…Randomize the KOD DNA polymerase gene using your method of choice (Chronopoulou & Labrou, 2011;Meyer, Ellefson, & Ellington, 2014;Wilson & Keefe, 2001) and use the Golden Gate cloning method (Engler et al, 2008) (or any other method of choice) to insert the library into the destination vector pTet-BsaI (or a destination vector of choice). Randomize the KOD DNA polymerase gene using your method of choice (Chronopoulou & Labrou, 2011;Meyer, Ellefson, & Ellington, 2014;Wilson & Keefe, 2001) and use the Golden Gate cloning method (Engler et al, 2008) (or any other method of choice) to insert the library into the destination vector pTet-BsaI (or a destination vector of choice).…”

Section: Library Transformation and Expressionmentioning

confidence: 99%

“…Moreover, the initial starting diversity can be further expanded in later rounds of selection via mutagenic PCR or DNA shuffling (Meyer et al, 2014;Wilson & Keefe, 2001). However, libraries of this size can be readily plumbed for function, assuming that they are designed properly.…”

Section: Csr For Polymerase Engineeringmentioning

confidence: 99%

“…One prominent example of recombinatorial mutagenesis is gene shuffling (Meyer et al, 2014), which relies on DNase I digestion and primerless gene reassembly by PCR to recombine one to many different DNA sequences in vitro at homologous crossover points. One prominent example of recombinatorial mutagenesis is gene shuffling (Meyer et al, 2014), which relies on DNase I digestion and primerless gene reassembly by PCR to recombine one to many different DNA sequences in vitro at homologous crossover points.…”

Section: Library Designmentioning

confidence: 99%

“…1. Randomize the KOD DNA polymerase gene using your method of choice (Chronopoulou & Labrou, 2011;Meyer, Ellefson, & Ellington, 2014;Wilson & Keefe, 2001) and use the Golden Gate cloning method (Engler et al, 2008) (or any other method of choice) to insert the library into the destination vector pTet-BsaI (or a destination vector of choice). After the ligation is complete, column-purify (using a kit such as DNA Clean & Concentrator-5 kit) 1 μg (estimated from vector DNA concentration) of the ligation and elute in 12 μl of ultrapure water.…”

Section: Library Transformation and Expressionmentioning

confidence: 99%

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Compartmentalized Self‐Replication for Evolution of a DNA Polymerase

Abil

Ellington

2018

CP Chemical Biology

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Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. E. coli host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR. CSR is a powerful method for engineering of polymerases since it allows selection under a variety of conditions, including the use of non-standard substrates. In this unit, we provide a step-by-step procedure for the selection of polymerases, using as an example the selection of reverse transcriptase activity starting from a library of Thermococcus kodakaraensis (KOD) DNA polymerase variants. © 2018 by John Wiley & Sons, Inc.

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