Tn5 Transposition Research Articles

Mobile-CRISPRi protocol optimization for Vibrionaceae.

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl β-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

Spatially resolved epigenome sequencing via Tn5 transposition and deterministic DNA barcoding in tissue.

Spatial epigenetic mapping of tissues enables the study of gene regulation programs and cellular functions with the dependency on their local tissue environment. Here we outline a complete procedure for two spatial epigenomic profiling methods: spatially resolved genome-wide profiling of histone modifications using in situ cleavage under targets and tagmentation (CUT&Tag) chemistry (spatial-CUT&Tag) and transposase-accessible chromatin sequencing (spatial-ATAC-sequencing) for chromatin accessibility. Both assays utilize in-tissue Tn5 transposition to recognize genomic DNA loci followed by microfluidic deterministic barcoding to incorporate spatial address codes. Furthermore, these two methods do not necessitate prior knowledge of the transcription or epigenetic markers for a given tissue or cell type but permit genome-wide unbiased profiling pixel-by-pixel at the 10 μm pixel size level and single-base resolution. To support the widespread adaptation of these methods, details are provided in five general steps: (1) sample preparation; (2) Tn5 transposition in spatial-ATAC-sequencing or antibody-controlled pA-Tn5 tagmentation in CUT&Tag; (3) library preparation; (4) next-generation sequencing; and (5) data analysis using our customed pipelines available at: https://github.com/dyxmvp/Spatial_ATAC-seq and https://github.com/dyxmvp/spatial-CUT-Tag . The whole procedure can be completed on four samples in 2-3 days. Familiarity with basic molecular biology and bioinformatics skills with access to a high-performance computing environment are required. A rudimentary understanding of pathology and specimen sectioning, as well as deterministic barcoding in tissue-specific skills (e.g., design of a multiparameter barcode panel and creation of microfluidic devices), are also advantageous. In this protocol, we mainly focus on spatial profiling of tissue region-specific epigenetic landscapes in mouse embryos and mouse brains using spatial-ATAC-sequencing and spatial-CUT&Tag, but these methods can be used for other species with no need for species-specific probe design.

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

Direct transposition of native DNA for sensitive multimodal single-molecule sequencing

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

An easily modifiable conjugative plasmid for studying horizontal gene transfer

Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e.g., fluorescent proteins) or antibiotic resistance genes would be efficient and convenient. Here, we introduced a single attTn7 site into the conjugative broad-host-range IncP-1 plasmid pKJK5 in a non-disruptive manner. Furthermore, a version with lower transfer rate and a non-conjugative version of pKJK5-attTn7 were also constructed. The advantage of having the attTn7 sites is that genes of interest can be introduced in a single step with very high success rate using the Tn7 transposition system. In addition, larger genetic fragments can be inserted. To illustrate the efficacy of the constructed pKJK5 plasmids, they were complemented with sfGFP (a gene encoding superfolder green fluorescent protein) in addition to seven different β-lactamase genes representing the four known classes of β-lactamases.

Spatial profiling of chromatin accessibility in mouse and human tissues

Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2–5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.

Structural basis of transposon end recognition explains central features of Tn7 transposition systems

SUMMARYTn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.

Enhanced CoQ10 production by genome modification of Rhodobacter sphaeroides via Tn7 transposition.

As a native CoQ10 producer, Rhodobacter sphaeroides has been extensively engineered to enhance CoQ10 production. However, the genetic manipulations using plasmids suffer from risk of plasmid loss during propagation process, biomass impairment due to cellular burden and bio-safety concerns. In this paper, genomic manipulations via Tn7 transposition was conducted to boost the CoQ10 biosynthesis in R. sphaeroides. The titer production and content of CoQ10 were improved by 18.44% and 18.87%, respectively compared to the wild type, when an additional copy of dxs and dxr were integrated into the genome. Further overexpression of idi and ispD by genomic integration created strain RSPCDDII with CoQ10 production and content of 81.23mg/L and 5.93mg/g, which were 54.28 and 55.97% higher than those of the wild type. The gene segments were successfully inserted into the attTn7 site of the R. sphaeroides genome. Meanwhile, the biomass was not affected. Compared to overexpression of genes on plasmids, this strategy could enhance protein expression to a proper level without affecting cell growth, and in a more stable manner.

Reduction of the Bacterial Genome by Transposon-Mediated Random Deletion.

Genome reduction is an important strategy in synthetic biology for constructing functional chassis cells or minimal genomes. However, the limited knowledge of complex gene functions and interactions makes genome reduction by rational design encounter a bottleneck. Here, we present an iterative and random genome reduction method for Escherichia coli, named "transposon-mediated random deletion (TMRD)". TMRD generates random double-strand breaks (DSBs) in the genome by combining Tn5 transposition with the CRISPR/Cas9 system and allows genomic deletions of various sizes at random positions during DSB repair through the intracellular alternative end-joining mechanism. Using E. coli MG1655 as the original strain, a pool of cells with multiple random genomic deletions were obtained after five reduction cycles. The growth rates of the obtained cells were comparable to that of MG1655, while the electroporation efficiency increased by at least 2 magnitudes. TMRD can generate a small E. coli library carrying multiple and random genomic deletions while enriching the cells with environmental fitness in the population. TMRD has the potential to be widely applied in the construction of minimal genomes or chassis cells for metabolic engineering.

Dual detection of chromatin accessibility and DNA methylation using ATAC-Me.

The epigenome is multidimensional, with individual molecular components operating on different levels to control transcriptional output. Techniques that combine measurements of these features can reveal their precise correspondence in genomic space, or temporal connectivity, to better understand how they jointly regulate genes. ATAC-Me is an integrated method to probe DNA methylation and chromatin accessibility from a single DNA fragment library. Intact nuclei undergo Tn5 transposition to isolate DNA fragments within nucleosome-free regions. Isolated fragments are exposed to sodium bisulfite before library amplification and sequencing. A typical ATAC-Me experiment detects ~60,000-75,000 peak regions (P < 0.05), covering ~3-4 million CpG sites with at least 5× coverage. These sites display a range of methylation values depending on the cellular and genomic context. The approach is well suited for time course studies that aim to capture chromatin and DNA methylation dynamics in tandem during cellular differentiation. The protocol is completed in 2 d with standard molecular biology equipment and expertise. Analysis of resulting data uses publicly available software requiring basic bioinformatics skills to interpret results.

Homologous Recombination Offers Advantages over Transposition-Based Systems to Generate Recombinant Baculovirus for Adeno-Associated Viral Vector Production.

Viral vectors have a great potential for gene delivery, but manufacturing is a big challenge for the industry. The baculovirus-insect cell is one of the most scalable platforms to produce recombinant adeno-associated virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and suffers technical constraints. Moreover, baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raises concerns about safety due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, a faster and safer method based on homologous recombination (HR) is investigated. First, the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes are confirmed. Strikingly, it is found that the exogenous cassette showed increased stability over passages when using the HR system. Finally, both materials generated high rAAV vector genome titers, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for its manufacturing. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products.

GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system

BackgroundRecombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as “GoldenBac”.ResultsThis method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2–15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein.ConclusionsThis robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.

MDR Salmonella enterica serovar Typhimurium ST34 carrying mcr-1 isolated from cases of bloodstream and intestinal infection in children in China

Children are vulnerable to Salmonella infection due to their immature immune system. Cases of infection with mcr-1-harbouring Salmonella in child inpatients have not been reported in China before. Salmonella isolates from gastroenteritis and bacteraemia were screened using primers targeting mcr-1. Complete genome sequences of mcr-1-harbouring isolates were determined using the PacBio RS II platform. The transferability of mcr-1-harbouring plasmids was verified by conjugation. We investigated two mcr-1-carrying polymyxin-resistant Salmonella enterica serovar Typhimurium ST34 isolates, S61394 and S44712, from bloodstream and intestinal Salmonella infection of two child inpatients, respectively. Both isolates were non-susceptible to commonly used antibiotics for children that compromised the success of clinical treatment and infection control. The mcr-1-harbouring plasmids pLS61394-MCR and pLS44712-MCR (from S61394 and S44712, respectively) were both conjugative pHNSHP45-2-like IncHI2-type epidemic plasmids carrying multiple resistance genes. Compared with pHNSHP45-2, a ∼33 kb insertion region encoding Tn7 transposition protein and heavy metal resistance proteins was identified in pLS61394-MCR, which might enhance adaptation of bacteria carrying this plasmid to various ecological niches. The phylogenetic tree of worldwide mcr-harbouring Salmonella indicated a host preference of mcr and a worldwide and cross-sectoral prevalence of the mcr-positive Salmonella ST34 clone. To our knowledge, for the first time we report completed whole genomes of mcr-1-positive MDR Salmonella Typhimurium ST34 isolated from infected children in China, suggesting that improved surveillance is imperative for tackling the dissemination of mcr-harbouring MDR Salmonella Typhimurium ST34.

Beyond adaptive immunity

CRISPR Biology Prokaryotic CRISPR-Cas systems defend bacterial cells from phage and plasmid infection. Strecker et al. characterized a CRISPR-Cas system that functions beyond adaptive immunity (see the Perspective by Hou and Zhang). Type V-K CRISPR-Cas from cyanobacteria was associated with a Tn7-like transposon and a natural nuclease–deficient effector Cas12k. Cas12k directed the insertion of Tn7-like transposons into target sites via RNA-guided Tn7 transposition. This system was reprogrammed to efficiently and specifically insert DNA both in vitro and into the Escherichia coli genome. Science , this issue p. [48][1]; see also p. [25][2] [1]: /lookup/doi/10.1126/science.aax9181 [2]: /lookup/doi/10.1126/science.aay2056

RNA-guided DNA insertion with CRISPR-associated transposases.

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

A new single-step protocol for rapid baculovirus-driven protein production in insect cells

BackgroundIn the last three decades, the Baculovirus expression vector system (BEV) has evolved to one of the most widely used eukaryotic systems for heterologous protein expression including approved vaccines and therapies. Despite the significant improvements introduced during the past years, the BEV system still has major drawbacks, primarily the time required to generate recombinant virus and virus instability for certain target proteins. In this study we show that the conventional method to generate recombinant Baculovirus using a Tn7 transposition based system can be shortened to a single-step transfection-only procedure without further amplification.MethodsIn a first step we have adapted a recently published protocol that replaces the standard liposome-based transfection procedure of adherent insect cells by transfecting insect cells in suspension with a preformed DNA-PEI complex generating P0 virus. We have then expressed and purified six different target proteins, among them four intracellular and two secreted proteins, by infecting insect cells either with P0 or P1 virus.ResultsWe demonstrate that transfection in suspension is as efficient as the standard protocol, but in addition allows generation of high amounts of P0 virus early in the process. To test if this P0 virus generated by bacmid transfection can be used directly for protein expression in either the screening or production process, we compared P0 versus amplified P1 virus-mediated protein expression. We show that protein expression levels, purity and yield of the purified proteins are equally high for P0 and P1.ConclusionThe standard protocol for generating recombinant baculovirus comprises transfection of the bacmid followed by one or two subsequent virus amplification steps. In this study we show that Baculovirus generated by transfection-only is equally efficient in driving protein expression. This reduces the time from bacmid DNA to protein to eight days and reduces the risk of virus decay. In contrast to transient gene expression protocols, the required amount of DNA is minimal: 100 µg bacmid DNA is sufficient for a production scale of 10 L.

Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI).

Single-cell genomics is important for biology and medicine. However, current whole-genome amplification (WGA) methods are limited by low accuracy of copy-number variation (CNV) detection and low amplification fidelity. Here we report an improved single-cell WGA method, Linear Amplification via Transposon Insertion (LIANTI), which outperforms existing methods, enabling micro-CNV detection with kilobase resolution. This allowed direct observation of stochastic firing of DNA replication origins, which differs from cell to cell. We also show that the predominant cytosine-to-thymine mutations observed in single-cell genomics often arise from the artifact of cytosine deamination upon cell lysis. However, identifying single-nucleotide variations (SNVs) can be accomplished by sequencing kindred cells. We determined the spectrum of SNVs in a single human cell after ultraviolet radiation, revealing their nonrandom genome-wide distribution.

Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems.

In the baculovirus shuttle vector (bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome, and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive (ts) plasmid, pSIM6. Using the resulting ts helper plasmid pMON7124(ts) and the ts donor plasmid pFB1(ts)-PH-GFP, a recombinant bacmid, bAcWT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For bAcWT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.

Monitoring survival of phenanthrene-utilizing Sphingobium sp. P2 in soil microcosms using green fluorescent protein as a marker

Phenanthrene is a polycyclic aromatic hydrocarbon which belongs to a class of toxic environmental pollutants that has been accumulating due to a variety of anthropogenic activities. We attempted to use Sphingobium sp. P2, which is capable of utilizing phenanthrene as its sole source of carbon and energy, in bioaugmentation treatments. For successful bioremediation, monitoring the survival and metabolic activity of bioaugmented bacteria are required. This study aimed to use green fluorescent protein as a marker to estimate the survival of Sphingobiumsp. P2 in bioaugmented soil microcosms. The gfp was integrated via Tn5 transposition into the chromosome of strain P2. The gfp-inserted strains were identified by green fluorescence emission under UV light. The gfp was stably maintained in strain P2, and the strain still retained the ability to use phenanthrene as a sole carbon and energy source. Soil microcosm experiments revealed that survival of the strain P2-gfp and its phenanthrene-degrading capacity may depend on indigenous microorganisms and nutrients in the soils. The strain P2-gfp can be used to evaluate the success of bioaugmentation.