“Cre/loxP plus BAC”: a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens

Hu, Shengbiao; Liu, Zhengqiang; Zhang, Xu; Zhang, Guoyong; Xie, Yi; Ding, Xuezhi; Mo, Xiaoyang; Stewart, A. Francis; Fu, Jun; Zhang, Youming; Xia, Liqiu

doi:10.1038/srep29087

Cited by 11 publications

(14 citation statements)

References 41 publications

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“…The modularity and orthogonality of the functional elements of the newly designed EXIT circuit means that this circuit may be readily applicable to a wide variety of plasmids with different replication origins. To confirm this, the EXIT circuit was assembled in other plasmid types with different replication origins, and four plasmids (pEC105, pEC102, pEC103 and pEC104) were constructed from pBelobac11 (single copy) [ 45 ], pBR322 (medium copy) [ 46 ], colE1 (high copy) [ 47 ] and pMB1 (very high copy) [ 48 ], respectively. The elimination efficiencies of these plasmids were determined (Fig.…”

Section: Resultsmentioning

confidence: 99%

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit

2017

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BackgroundPlasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation.ResultsTo bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I-SceI and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing.ConclusionsOur novel EXIT circuit, which exploits the homing endonuclease I-SceI, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-017-0072-5) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit

2017

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“…Napsamycin, daptomycin (Du et al, 2015) Siderophore (Hu et al, 2016) Erythromycin (Dai et al, 2015) iCatch • Time-consuming;…”

Section: Llhrmentioning

confidence: 99%

“…Similarily, iCatch intergrates homing endonucleases I-SceI and PI-PspI recognition sites flanking the region of interest, after which the genome is isolated and digested with I-SceI or PI-PspI and then self-ligated to clone the target BGC in vitro (Wang et al, 2019). Moreover, several groups have developed methods that express recombinases to extract DNA fragments between two integrase recognition sites and circularize the plasmid in vivo (Figure 1D), such as phage φBT1 integrase-mediated site-specific recombination (Du et al, 2015), Cre/loxP plus BAC (Hu et al, 2016). These plasmids can then be isolated from the native host for heterologous expression.…”

Section: Cloning Strategiesmentioning

confidence: 99%

Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters

Lin

Nielsen

Liu

2020

Front. Bioeng. Biotechnol.

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Since the discovery of penicillin, natural products and their derivatives have been a valuable resource for drug discovery. With recent development of genome mining approaches in the post-genome era, a great number of natural product biosynthetic gene clusters (BGCs) have been identified and these can potentially be exploited for the discovery of novel natural products that can find application as pharmaceuticals. Since many BGCs are silent or do not express in native hosts under laboratory conditions, heterologous expression of BGCs in genetically tractable hosts becomes an attractive route to activate these BGCs to discover the corresponding products. Here, we highlight recent achievements in cloning and discovery of natural product biosynthetic pathways via intact BGC capturing, and discuss the prospects of high-throughput and multiplexed cloning of rational-designed gene clusters in the future.

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“…For example, Hu and coworkers developed a Cre/loxP based method [47]. The loxP sites are first integrated into the boundary regions of the target BGC with elements that are necessary for plasmid replication.…”

Section: Activation Of Silent Bgcs In Heterologous Hostsmentioning

confidence: 99%

Breaking the silence: new strategies for discovering novel natural products

Ren

Wang

Zhao

2017

Current Opinion in Biotechnology

103

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Natural products have been a prolific source of antibacterial and anticancer drugs for decades. One of the major challenges in natural product discovery is that the vast majority of natural product biosynthetic gene clusters (BGCs) have not been characterized, partially due to the fact that they are either transcriptionally silent or expressed at very low levels under standard laboratory conditions. Here we describe the strategies developed in recent years (mostly between 2014–2016) for activating silent BGCs. These strategies can be broadly divided into two categories: approaches in native hosts and approaches in heterologous hosts. In addition, we briefly discuss recent advances in developing new computational tools for identification and characterization of BGCs and high-throughput methods for detection of natural products.

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“Cre/loxP plus BAC”: a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens

Cited by 11 publications

References 41 publications

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit

Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters

Breaking the silence: new strategies for discovering novel natural products

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