Designing and Implementing Algorithmic DNA Assembly Pipelines for Multi-Gene Systems

Hsu, Szu Yi; Smanski, Michael J.

doi:10.1007/978-1-4939-7295-1_9

Cited by 5 publications

(8 citation statements)

References 23 publications

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“…To minimize the confounding effects of medium composition on small molecule titers/yield, we only included used cis-regulatory elements that gave consistent expression of a reporter gene in two growth media, ISM3 and PCNM (Figure 1d and Figure S4). Combining these natural and synthetic genetic elements using one-pot type IIS restriction digestion/ligation reactions, 25 an initial set of seven synthetic gene clusters (SGCs) were produced (Figure 1e). We opted for an iterative DNA assembly pipeline comprising modest (4−5 part) assembly reactions to keep the efficiency high at each step.…”

Section: ■ Resultsmentioning

confidence: 99%

Semisynthesis of the Neuroprotective Metabolite, Serofendic Acid

et al. 2019

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Serofendic acid is a natural neuroprotective molecule found in fetal calf serum. It is able to protect neurons against mechanisms of cell death associated with neurodegenerative disease. Because only trace quantities are present in fetal calf serum and complete chemical syntheses are long and inefficient, its development as a therapeutic agent has been slow. We engineered a heterologous metabolic pathway in Streptomyces to produce a late-stage synthetic intermediate, ent-atiserenoic acid, at high titers. We completed the total synthesis of serofendic acid from this intermediate in four steps.

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Section: ■ Resultsmentioning

confidence: 99%

Semisynthesis of the Neuroprotective Metabolite, Serofendic Acid

et al. 2019

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“…Our results indicated that Nimble Cloning is not only more convenient than Gibson assembly, but it also results in a higher cloning efficiency (Figure 2A). Previous studies confirmed that Golden Gate cloning, which also involves a one-step digestion/ligation reaction, is more efficient than traditional cloning methods requiring a two-step digestion and ligation reaction (Engler et al, 2009;Hsu and Smanski, 2018). The increased efficiency of onestep cloning may be due to the decrease in the number of steps involving the DNA.…”

Section: Discussion the Advantages Of Nimble Cloning Over Other Methodsmentioning

confidence: 99%

Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning

Zeng

Shen

et al. 2020

Front. Bioeng. Biotechnol.

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Molecular cloning is one of the most fundamental technologies in molecular biology, and has been critical for driving biotechnological advances. In this study, we have developed a novel method for standardized molecular cloning. The cloning technique known as "Nimble Cloning" uses the restriction enzyme, SfiI, in combination with the T5 exonuclease, to linearize the vector and generate 3 ′-overhangs simultaneously. Both PCR products and plasmids can be used for the cloning reaction in the Nimble Cloning system. The cloning system is highly efficient, suitable for gene expression in both prokaryotic and eukaryotic expression systems, and enables the reuse of DNA fragments or plasmid entry clones. Nimble Cloning is applicable for the cloning of single or multiple fragments, as well as multi-site cloning. Due also to its simplicity and versatility, the cloning method has great potential for the modular assembly of DNA constructs.

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“…pCDS was constructed via with a one-pot Golden Gate assembly as previously described using 5 U Bbs I (New England Biolabs, R0539S) and 5 U T4 Ligase (Promega, # M1804) from two PCR fragments. The origin of replication and selectable Kanamycin marker was amplified using oligonucleotide primers (pCDS_vecF, pCDS_vecR) from plasmid pMJS1AE and lacZα gene product was amplified from plasmid pMJS1AE using oligonucleotide primers (pCDS_lacZF, pCDS_lacZR) that contain lacZ-specific sequences, a Sap I recognition site, 4-bp assembly scar sequence, and an Aar I recognition site.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…CDSs were synthesized by Twist Bioscience (San Francisco, CA) with (5′-atgcaCACCTGCTACTA-) and (-TATGGGCAGGTGatgca-3′) appended to the 5′ and 3′ ends, respectively, to enable modular cloning. CDSs were cloned into the pCDS vector via a one-pot Aar I restriction digestion-ligation reaction (GeneArt Type IIs Assembly Kit, Aar I ThermoFisher Scientific, # A15916) …”

Section: Materials and Methodsmentioning

confidence: 99%

Model-Driven Engineering of N-Linked Glycosylation in Chinese Hamster Ovary Cells

et al. 2019

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Chinese hamster ovary (CHO) cells are used for industrial production of protein-based therapeutics (i.e., "biologics"). Here we describe a method for combining systems-level kinetic models with a synthetic biology platform for multigene overexpression to rationally perturb N-linked glycosylation. Specifically, we sought to increase galactose incorporation on a secreted Immunoglobulin G (IgG) protein. We rationally design, build, and test a total of 23 transgenic cell pools that express single or three-gene glycoengineering cassettes comprising a total of 100 kilobases of engineered DNA sequence. Through iterative engineering and model refinement, we rationally increase the fraction of bigalactosylated glycans five-fold from 11.9% to 61.9% and simultaneously decrease the glycan heterogeneity on the secreted IgG. Our approach allows for rapid hypothesis testing and identification of synergistic behavior from genetic perturbations by bridging systems and synthetic biology.

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Designing and Implementing Algorithmic DNA Assembly Pipelines for Multi-Gene Systems

Cited by 5 publications

References 23 publications

Semisynthesis of the Neuroprotective Metabolite, Serofendic Acid

Semisynthesis of the Neuroprotective Metabolite, Serofendic Acid

Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning

Model-Driven Engineering of N-Linked Glycosylation in Chinese Hamster Ovary Cells

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