USER-Derived Cloning Methods and Their Primer Design

Salomonsen, Bo; Mortensen, Uffe Hasbro; Halkier, Barbara Ann

doi:10.1007/978-1-62703-764-8_5

Cited by 14 publications

(11 citation statements)

References 32 publications

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“…Uracil-specific excision reagent (USER) cloning [21] was used to introduce DNA sequences encoding fluorescent moieties and affinity tags to rat Abcc6 (rAbcc6) in the Gateway entry vector pEntr223 [7]. cDNA sequences were amplified using Phusion U PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA).…”

Section: Generation Of Constructs Encoding Fluorescent Rat Abcc6 Fusimentioning

confidence: 99%

Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology

et al. 2020

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ABCC6 mediates release of ATP from hepatocytes into the blood. Extracellularly, ATP is converted into the mineralization inhibitor pyrophosphate. Consequently, inactivating mutations in ABCC6 give low plasma pyrophosphate and underlie the ectopic mineralization disorder pseudoxanthoma elasticum. How ABCC6 mediates cellular ATP release is still unknown. Fluorescent ABCC6 fusion proteins would allow mechanistic studies, but fluorophores attached to the ABCC6 Nor C-terminus result in intracellular retention and degradation. Here we describe that intramolecular introduction of fluorophores yields fully functional ABCC6 fusion proteins. A corresponding ABCC6 variant in which the catalytic glutamate of the second nucleotide binding domain was mutated, correctly routed to the plasma membrane but was inactive. Finally, N-terminal His 10 or FLAG tags did not affect activity of the fusion proteins, allowing their purification for biochemical characterization.

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Section: Generation Of Constructs Encoding Fluorescent Rat Abcc6 Fusimentioning

confidence: 99%

Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology

et al. 2020

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“…The pMCSG7 vector contains an N-terminal 6xHis tag followed by a TEV cleavage site. USER cloning [41] was employed to clone KCTD6 BTB (residues 10-115) into the pET-2ST vector, which contains an N-terminal SUMO fusion protein and 6xHis tag followed by a TEV cleavage site. KCTD5 FL (residues 1-234) was cloned into the PSJ5 vector by restriction enzyme cloning.…”

Section: Cloning Protein Expression and Purificationmentioning

confidence: 99%

Structural Insights into KCTD Protein Assembly and Cullin3 Recognition

Chu

Nielsen

et al. 2016

Journal of Molecular Biology

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Cullin3 (Cul3)-based ubiquitin E3 ligase complexes catalyze the transfer of ubiquitin from an E2 enzyme to target substrate proteins. In these assemblies, the C-terminal region of Cul3 binds Rbx1/E2-ubiquitin, while the N-terminal region interacts with various BTB (bric-à-brac, tramtrack, broad complex) domain proteins that serve as substrate adaptors. Previous crystal structures of the homodimeric BTB proteins KLHL3, KLHL11 and SPOP in complex with the N-terminal domain of Cul3 revealed the features required for Cul3 recognition in these proteins. A second class of BTB-domain-containing proteins, the KCTD proteins, is also Cul3 substrate adaptors, but these do not share many of the previously identified determinants for Cul3 binding. We report the pentameric crystal structures of the KCTD1 and KCTD9 BTB domains and identify plasticity in the KCTD1 rings. We find that the KCTD proteins 5, 6, 9 and 17 bind to Cul3 with high affinity, while the KCTD proteins 1 and 16 do not have detectable binding. Finally, we confirm the 5:5 assembly of KCTD9/Cul3 complexes by cryo-electron microscopy and provide a molecular rationale for BTB-mediated Cul3 binding specificity in the KCTD family.

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“…Among the various in vitro DNA editing methods, such as SLIC (M. Z. Li & Elledge, ), USER (Salomonsen, Mortensen, & Halkier, ), SSRTA (Zhang, Zhao, & Ding, ) and SIRA (Colloms et al, ), the low efficiency at certain conditions or the special requirements for certain applications limit their wide utilization. Therefore, more versatile DNA editing/cloning methods were developed, such as Type IIs RE based Golden Gate assembly and PfAgo mediated PfAgo/AREs platform (Enghiad & Zhao, ; Engler et al, ).…”

Section: Discussionmentioning

confidence: 99%

“…Among the various in vitro DNA editing methods, such as SLIC (M. Z. Li & Elledge, 2012), USER (Salomonsen, Mortensen, & Halkier, 2014), SSRTA (Zhang, Zhao, & Ding, 2011) and SIRA (Colloms et al, 2014), the low efficiency at certain conditions or the special requirements for certain applications limit their wide utilization.…”

Section: Discussionmentioning

confidence: 99%

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

Wang

Liu

et al. 2019

Biotech & Bioengineering

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As the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a (previously known as Cpf1) system cleaves double-stranded DNA and produces a sticky end, it could serve as a useful tool for DNA assembly/editing. To broaden its application, a variety of engineered FnCas12a proteins are generated with expanded protospacer adjacent motif (PAM) requirements. Two variants (FnCas12a-EP15 and EP16) increased the targeting range of FnCas12a by approximately fourfold. They can efficiently recognize a broad range of PAM sequences including YN (Y = C or T), TAC and CAA. Meanwhile, based on our demonstration that FnCas12a is active from 16 to 60°C, we developed an "improved CRISPR-Cas12a-assisted one-pot DNA editing" (iCOPE) method to facilitate DNA editing by combining the crRNA transcription, digestion, and ligation in one pot. By applying iCOPE, the editing efficiency reached 72-100% for two DNA fragment assemblies, and for the 21 kb large DNA construct modification, the editing efficiency can reach 100%. Thanks to the advantages of Cas12a, iCOPE with only one digestion enzyme could replace current a variety of restriction enzymes to perform the cloning in one pot with almost no sequence constraints. Taken together, this study offers an expanded DNA targeting scope of CRISPR systems and could serve as an efficient seamless one-pot DNA editing tool. K E Y W O R D SCas12a variants, DNA editing, FnCas12a, one pot, synthetic biology

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USER-Derived Cloning Methods and Their Primer Design

Cited by 14 publications

References 32 publications

Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology

Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology

Structural Insights into KCTD Protein Assembly and Cullin3 Recognition

Improved CRISPR‐Cas12a‐assisted one‐pot DNA editing method enables seamless DNA editing

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