FIG 1 - uploaded by Andrew Tolonen
Content may be subject to copyright.
Overview of genomic deletion and insertion in C. phytofermentans. (A) pQlox71 is introduced for genomic insertion of a lox71 (L71) site using the LtrA protein encoded by the targetron. (B) pQlox71 is cured and pQlox66 is introduced for genomic insertion of a lox66 (L66) site. (C) pQlox66 is cured, and pQcre1 is introduced for Cre-mediated recombination to delete the sequence between the lox66 and lox71 sites. (D) In the resulting strain, the deletion and lox72 site are confirmed by PCR (arrows show primers). (E) pQadd1 is introduced for genomic delivery of a lox511/71 (L5-71) and loxFAS/66 (LF-66) cassette into the genome. (F) pQadd1 is cured and pQadd2 is introduced, bearing the desired insertion sequence flanked by lox511/66 (L5-66) and loxFAS/71 (LF-71) sites. (G) pQcre2 is introduced for Cre-mediated RMCE. (H) The resulting strain has a genomic copy of the insert sequence flanked by lox511/72 (L5-72) and loxFAS/72 (LF-72) sites in the genome, which is confirmed by PCR (arrows show primers).
Source publication
Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply th...
Contexts in source publication
Context 1
... we describe a way to make large precise deletions ( Fig. 1A to D) and insertions ( Fig. 1E to H) in clostridial genomes using C. phytofermentans as a model. Targetrons and Cre recombinase have been used together to make genomic insertions in Escherichia coli and deletions in E. coli and Staphylococcus aureus (13), showing that these tools can be used together in Gram-negative and Gram-positive ...
Context 2
... we describe a way to make large precise deletions ( Fig. 1A to D) and insertions ( Fig. 1E to H) in clostridial genomes using C. phytofermentans as a model. Targetrons and Cre recombinase have been used together to make genomic insertions in Escherichia coli and deletions in E. coli and Staphylococcus aureus (13), showing that these tools can be used together in Gram-negative and Gram-positive bacteria. We developed a ...
Context 3
... for genomic deletion and insertion. Our strategy to make targeted deletions (Fig. 1A to D) and insertions ( Fig. 1E to H) in the C. phytofermentans genome is based on sequential introduction and removal of conditionally replicating plasmids carrying lox sites or Cre recombinase. We constructed plasmids for targetron-mediated delivery of lox sites by inserting the lox cassettes into the unique MluI site in domain IV of ...
Context 4
... for genomic deletion and insertion. Our strategy to make targeted deletions (Fig. 1A to D) and insertions ( Fig. 1E to H) in the C. phytofermentans genome is based on sequential introduction and removal of conditionally replicating plasmids carrying lox sites or Cre recombinase. We constructed plasmids for targetron-mediated delivery of lox sites by inserting the lox cassettes into the unique MluI site in domain IV of the intron, a site that supports DNA ...
Context 5
... the lox cassettes into the unique MluI site in domain IV of the intron, a site that supports DNA insertions while retaining targetron activity (13). Targetrons can be programmed to insert into the genome in either orientation, but both lox linkers must be in the same relative orientation in the genome for Cre to delete the intervening region (Fig. 1C). Similarly, RMCE requires two Cre-based recombinations between tandem lox sites whose orientation determines that of the inserted DNA ( Fig. 1E to H). We thus constructed lox-containing targetrons in pQint (10) with either orientation of lox sites: lox71 (pQlox71F and pQlox71R) and lox66 (pQlox66F and pQlox66R) for genome deletions and ...
Context 6
... (13). Targetrons can be programmed to insert into the genome in either orientation, but both lox linkers must be in the same relative orientation in the genome for Cre to delete the intervening region (Fig. 1C). Similarly, RMCE requires two Cre-based recombinations between tandem lox sites whose orientation determines that of the inserted DNA ( Fig. 1E to H). We thus constructed lox-containing targetrons in pQint (10) with either orientation of lox sites: lox71 (pQlox71F and pQlox71R) and lox66 (pQlox66F and pQlox66R) for genome deletions and a tandem lox511/71-loxFAS/66 cassette (pQadd1F and pQadd1R) for genome insertion by RMCE (Table 1; see also Fig. S1 in the supplemental ...
Context 7
... determines that of the inserted DNA ( Fig. 1E to H). We thus constructed lox-containing targetrons in pQint (10) with either orientation of lox sites: lox71 (pQlox71F and pQlox71R) and lox66 (pQlox66F and pQlox66R) for genome deletions and a tandem lox511/71-loxFAS/66 cassette (pQadd1F and pQadd1R) for genome insertion by RMCE (Table 1; see also Fig. S1 in the supplemental material). These plasmids all contain the erythromycin resistance gene from Streptococcus pneumoniae Tn1545 (21) for selection in E. coli and C. phytofermentans and the pAM1 origin that replicates stably in C. phytofermentans but can be cured by serial transfer in liquid medium lacking antibiotics (10). This ...
Context 8
... express Cre recombinase in C. phytofermentans for genomic deletion between lox71 and lox66 sites, we constructed pQcre1 ( Fig. S1; Table 1) in which cre is expressed from the Bacillus anthracis PpagA promoter, a well-characterized constitutive promoter that is widely functional in Gram-positive bacteria (22). Initially, we attempted to deliver a tandem lox cassette and cre on a single plasmid for RMCE but were unable to construct such a plasmid likely because of ...
Context 9
... genes unexpectedly decreased C. phytofermentans growth on nonpreferred carbon sources, and the extremely high upregulation of cphy2976 suggests that it plays an important role in cell fitness under these conditions. Genomic insertion by RMCE. To effectuate targeted genomic insertions in C. phytofermentans by RMCE, the targetron of pQadd1R (Fig. S1) was customized to insert tandem lox sites with incompatible linkers (lox511/71 and loxFAS/66) at 96 bp from the start of cphy1575 (Fig. 5A) to produce pQadd1R.1575. The cphy1575 gene is homolo- msphere.asm.org 6 gous to B. subtilis AprX, a nonessential S8 subtilisin serine protease that degrades heterologous protein in stationary ...
Context 10
... into cphy1575 by PCR (Fig. 5B), and cured the plasmid to yield strain int1575 (Table 1). We confirmed that int1575 did not contain any additional off-site targetron insertions by inverse PCR, which yielded only the expected 5.1-kb band corresponding to the insertion in cphy1575 (Fig. 5C). pQadd2 contains tandem lox511/66 and loxFAS/71 sites (Fig. S1) to facilitate RMCE with the complementary cassette integrated in the genome of strain int1575 (Fig. 5A). The lox sites in pQadd2 are separated by SpeI and XhoI sites into which we cloned a version of the FbFP oxygen-independent green fluorescent protein from Pseudomonas putida that has been codon optimized for clostridia (32). The ...
Similar publications
Background Salmonella enterica serovar Typhimurium persists as one of the most frequent food-borne zoonoses, causing a major public health concern worldwide. Furthermore, Salmonella infection has a large economic impact. Globally, the main sources of infection for humans include the consumption of contaminated poultry meat and eggs. In animals howe...
Citations
... Designed group II intron called targetrons enabled gene inactivation by targeted chromosome insertion in various Lachnospiraceae with efficiencies ranging from 12.5%-100% (Tolonen et al., 2009;Tolonen et al., 2015a;Cerisy et al., 2019a;Jin et al., 2022) (Figure 4D). Multi-gene fragments can be excised and inserted by modifying targetrons to deliver lox sites into the genome that act as anchor points for Cre-mediated recombination, which has been applied to delete a 39 kb prophage in L. phytofermentans (Cerisy et al., 2019b). ...
The Lachnospiraceae is a family of anaerobic bacteria in the class Clostridia with potential to advance the bio-economy and intestinal therapeutics. Some species of Lachnospiraceae metabolize abundant, low-cost feedstocks such as lignocellulose and carbon dioxide into value-added chemicals. Others are among the dominant species of the human colon and animal rumen, where they ferment dietary fiber to promote healthy gut and immune function. Here, we summarize recent studies of the physiology, cultivation, and genetics of Lachnospiraceae, highlighting their wide substrate utilization and metabolic products with industrial applications. We examine studies of these bacteria as Live Biotherapeutic Products (LBPs), focusing on in vivo disease models and clinical studies using them to treat infection, inflammation, metabolic syndrome, and cancer. We discuss key research areas including elucidation of intra-specific diversity and genetic modification of candidate strains that will facilitate the exploitation of Lachnospiraceae in industry and medicine.
... This strategy has been used to introduce lox sites to model and resident gut hosts where the intron containing the lox site acts as a "landing-pad" for a cargo vector containing complementary lox sites. Integration of the genetic cargo can then be incorporated into the target genome by expressing the Cre recombinase (62,63). Lox-based landing-pad sites have also successfully transferred genetic material to resident gut hosts when coupled with transposases, such as in the CRAGE system (64). ...
Techniques by which to genetically manipulate members of the microbiota enable both the evaluation of host-microbe interactions and an avenue by which to monitor and modulate human physiology. Genetic engineering applications have traditionally focused on model gut residents, such as Escherichia coli and lactic acid bacteria. However, emerging efforts by which to develop synthetic biology toolsets for "nonmodel" resident gut microbes could provide an improved foundation for microbiome engineering. As genome engineering tools come online, so too have novel applications for engineered gut microbes. Engineered resident gut bacteria facilitate investigations of the roles of microbes and their metabolites on host health and allow for potential live microbial biotherapeutics. Due to the rapid pace of discovery in this burgeoning field, this minireview highlights advancements in the genetic engineering of all resident gut microbes.
... 50 The results of this study provide a foundation for construction of genetic circuits with experimentally modulated gene expression in C. phytofermentans. These approaches complement previous technologies to study C. phytofermentans genetics using targetron-based gene inactivation, 30 large-scale genome insertion and deletion, 31 and in vivo directed evolution. 51 Cas12a could be used to make genomic changes in C. phytofermentans, as has been demonstrated in some other Clostridia. ...
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
... The cre/lox system has been used as a deletion system on many occasions, due to its ability to act in both prokaryotic and eukaryotic cells. In addition, the usage of mutant lox sites to facilitate deletions that result in an inactive lox has also been demonstrated in bacteria, such as being used to knock out single genes in series (Pan et al., 2011), or to knock out large but targeted genome region using either targetrons (Cerisy et al., 2019) or via recombineering (Xin et al., 2018). ...
The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non‐essential genes that could be removed simultaneously, and other non‐essential regions that could not, providing a guide for future genome reductions.
... In total, we found that 8/47 (17%) of papers surveyed had incorrectly labelled their lox sites. Of these, 7 of the 8 papers [17][18][19][20][21][22][23] had simply mislabelled the lox sites, ascribing the lox66 name to the canonical lox71, and vice versa. The remaining papers [24] correctly annotated the lox sites, but implied the directionality of the lox site was in the opposite direction to which it was written. ...
The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilized across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites. Two of the most commonly used mutant sites are named lox66 and lox71, which recombine to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardized naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae .
... The Cre/lox system has been used as a deletion system on many occasions, due to its ability to act in both prokaryotic and eukaryotic cells. In addition, the usage of mutant lox sites to facilitate deletions that result in an inactive lox has also been demonstrated in bacteria, such as being used to knock out single genes in series (Pan et al., 2011), or to knock out large but targeted genome region using either targetrons (Cerisy et al., 2019) or via recombineering (Xin et al., 2018). ...
The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and lox71, to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors, and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardised naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae .
Data Summary
The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.
Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.
DNA has been pursued as a novel biomaterial for digital data storage. While large-scale data storage and random access have been achieved in DNA oligonucleotide pools, repeated data accessing requires constant data replenishment, and these implementations are confined in professional facilities. Here, a mobile data storage system in the genome of the extremophile Halomonas bluephagenesis, which enables dual-mode storage, dynamic data maintenance, rapid readout, and robust recovery. The system relies on two key components: A versatile genetic toolbox for the integration of 10-100 kb scale synthetic DNA into H. bluephagenesis genome and an efficient error correction coding scheme targeting noisy nanopore sequencing reads. The storage and repeated retrieval of 5 KB data under non-laboratory conditions are demonstrated. The work highlights the potential of DNA data storage in domestic and field scenarios, and expands its application domain from archival data to frequently accessed data.
The sustainable production of solvents from above ground carbon is highly desired. Several clostridia naturally produce
solvents and use a variety of renewable and waste-derived substrates such as lignocellulosic biomass and gas mixtures
containing H2/CO2 or CO. To enable economically viable production of solvents and biofuels such as ethanol and butanol,
the high productivity of continuous bioprocesses is needed. While the frst industrial-scale gas fermentation facility operates continuously, the acetone–butanol–ethanol (ABE) fermentation is traditionally operated in batch mode. This review
highlights the benefts of continuous bioprocessing for solvent production and underlines the progress made towards its
establishment. Based on metabolic capabilities of solvent producing clostridia, we discuss recent advances in systems-level
understanding and genome engineering. On the process side, we focus on innovative fermentation methods and integrated
product recovery to overcome the limitations of the classical one-stage chemostat and give an overview of the current
industrial bioproduction of solvents.
