Exogenous isolation of conjugative plasmids from pesticide contaminated soil

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10?μg/ml) and tetracycline (20?μg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2?×?10(-6) and 9.2?×?10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.     

Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.     

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10⁻⁵ to 10⁻⁸ per recipient. A total of 12 distinct plasmids, designated pB1–pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10⁻¹ to 10⁻² per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPβ subgroup, whereas two plasmids were identified as IncPα plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPβ plasmids at the DNA sequence level. In order to characterize the gene “load” of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified. Received: 11 October 1999 / Accepted: 11 January 2000     

An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same

The Gateway^® recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.     

pEOC01: a plasmid from Pediococcus acidilactici which encodes an identical streptomycin resistance (aadE) gene to that found in Campylobacter jejuni

The complete nucleotide sequence of pEOC01, a plasmid (11,661 bp) from Pediococcus acidilactici NCIMB 6990 encoding resistance to clindamycin, erythromycin, and streptomycin was determined. The plasmid, which also replicates in Lactococcus and Lactobacillus species contains 16 putative open reading frames (ORFs), including regions annotated to encode replication, plasmid maintenance and multidrug resistance functions. Based on an analysis the plasmid replicates via a theta replicating mechanism closely related to those of many larger Streptococcus and Enterococcus plasmids. Interestingly, genes homologous to a toxin/antitoxin plasmid maintenance system are present and are highly similar to the omega-epsilon-zeta operon of Streptococcus plasmids. The plasmid contains two putative antibiotic resistance homologs, an ermB gene encoding erythromycin and clindamycin resistance, and a streptomycin resistance gene, aadE. Of particular note is the aadE gene which holds 100% identity to an aadE gene found in Campylobacter jejuni plasmid but which probably originated from a Gram-positive source. This observation is significant in that it provides evidence for recent horizontal transfer of streptomycin resistance from a lactic acid bacterium to a Gram-negative intestinal pathogen and as such infers a role for such plasmids for dissemination of antibiotic resistance genes possibly in the human gut.     

Cloning of the peacdc2 homologue by efficient immunological screening of PCR products

A homologue of the ubiquitous eukaryotic cell cycle regulatory gene,cdc2, has been cloned fromPisum sativum, the garden pea. A novel immunological strategy was devised and implemented for screening PCR products generated by degenerate oligonucleotide primers. We used PCR to construct a deletion derivative of anEscherichia coli expression plasmid carrying theSchizosaccharomyces pombe cdc2 gene. The deleted segment encoded the domain recognized by monoclonal antibody MAb-J4, a reagent which also detects a single protein in extracts of all plant species we have examined. PCR products, generated by appropriatecdc2 primers, were ligated into new restriction sites flanking the deletion, reconstituting the deleted epitope. This strategy, first validated on a cloned yeastcdc2 template as control, was applied to the highly efficient cloning of a cDNA segment comprising 60% of the peacdc2 homologue. DNA sequencing revealed strong amino acid sequence conservation among thecdc2 gene products from pea, yeast and animal cells. Genomic Southern analysis indicated that thecdc2 gene occurs as a single copy in pea. An additionalcdc2-like clone was recovered which displays amino acid sequence similarity with that of peacdc2. The reported cloning and screening strategy, though limited by the availability of appropriate immunological reagents, provides not only an efficient means of screening heterogeneous PCR products generated by degenerate probes and/or low stringency PCR, but also product verification by immunological criteria.     

EB病毒胸苷激酶基因的定向克隆

采用异硫氰酸胍（ＧｕＳＣＮ）和硅藻从Ｂ９５－８细胞中快速抽摸板ＤＮＡ。根据ＥＢ病毒（ＥＢＶ）Ｂ９５－８株ＤＮＡ全序列及编码ＥＢＶ胸苷激酶（ＴＫ）的开放读框ＢＸＬＦ１的结构,设计合成一对引物,并在引物的５′一端分别引入ＥｃｏＲＩ和ＰｓｔＩ切点,用ＰＣＲ技术扩增出一含完整的ＥＢＶＴＫ基因的１．８４３ＫｂＤＮＡ片段,ＮｃｏＩ酶切分析鉴定,ＥｃｏＲＩ／ＰｓｔＩ双酶切ＰＣＲ产物和载体,使目的基因定向克隆至选     

Plasmids in clinical isolates ofStreptococcus pneumoniae

Forty clinical isolates ofStreptococcus pneumoniae with various antibiotic resistance profiles were screened for the presence of plasmids. Plasmids were demonstrated in five isolates. Three procedures for plasmid isolation were evaluated. A 10.8-kb plasmid was demonstrated by all three methods, but a further four plasmids were detected with one method only. The sizes of these plasmids were 11.5 kb, 10.8 kb, and 3.0 kb (two strains). Curing experiments were performed, but no plasmid/antibiotic correlation was observed. Tetracycline, erythromycin, and clindamycin resistance was lost in one strain, although the plasmid was still present.     

杂合启动子HREAF的构建及其肝癌特异性分析

本研究将在几乎所有肝癌细胞中均有高特异性的杂合启动子HREAF用于构建肝癌特异性溶瘤腺病毒。根据献报道的缺氧应答元件(HRE)和人甲胎蛋白(AFP)核心启动子(AF0.3)基因序列,设计并合成2对引物,采用PCR方法从肝癌细胞基因组DNA中扩增获得大小分别约为560bp的HRE DNA片段和310bp的AF0.3DNA片段,连接到pGEM—T Easy载体进行测序,证明获得了HRE和AF0.3的基因片段。将HRE和AF0．3的PCR产物分别进行PstI和SspI酶切并末端补平后直接连接,将此约700bp的连接产物克隆到pGEM—T Easy载体进行测序,证明杂合启动子HREAF构建成功。将HREAF亚克隆至腺病毒穿梭载体pShuttle并在其后克隆入受其调控的腺病毒早期基因E1,构建成肝癌特异性溶瘤腺病毒穿梭载体pShuttle—HREAF—E1,经PCR及酶切鉴定。将pShuttle—HREAF—E1转染肺癌细胞株及AFP产量不等的不同人肝癌细胞株,经Ela的RT—PCR检测证实杂合启动子HREAF可调控目的基因在AFP产量不等的不同人肝癌细胞系中特异性高表达。杂合启动子HREAF及腺病毒穿梭载体pShuttle—HREAF—E1的构建为进一步研制肝癌特异性重组溶瘤腺病毒及其体内外肝癌特异杀伤作用的研究打下了基础。     

PCR-mediated deletion of plasmid DNA

The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.     

Experimental Evolution of Gene Duplicates in a Bacterial Plasmid Model

The fate of gene duplicates subjected to diversifying selection was tested experimentally in a bacterial system. The wild-type TEM-1 β-lactamase gene confers resistance to ampicillin but not to cefotaxime. Point mutations confer cefotaxime resistance, but they compromise ampicillin resistance. Thus, selection for both drug resistances in a bacterium with two copies of β-lactamase should favor the divergence of one copy to improve cefotaxime resistance while maintaining the other copy to preserve ampicillin resistance. This selection was performed on a bacterium with identical sequences of β-lactamase on two separate, compatible plasmids. As expected, one plasmid evolved increased cefotaxime resistance when appropriately strong cefotaxime selection was applied. However, the cefotaxime-resistant plasmid maintained sufficient ampicillin resistance to tolerate the concentration of ampicillin used, and the other plasmid was lost. Hosts carrying both the cefotaxime-resistant and wild-type plasmids were then subjected to various higher concentrations of both drugs to find conditions that would ensure the maintenance of both plasmids. In a striking contradiction to our model, no such conditions were found. The fitness cost of carrying both plasmids increased dramatically as antibiotic levels were raised, and either the wild-type plasmid was lost or the cells did not grow. This study highlights the importance of the cost of duplicate genes and the quantitative nature of the tradeoff in the evolution of gene duplication through functional divergence. Reviewing Editor: Dr. Margaret Riley     

Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli

A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error‐prone PCR or molecular shuffling, and a linear vector backbone was prepared by high‐fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap‐extension PCR with a pair of 5'‐phosphorylated primers. Third, full‐length linear plasmids with phosphorylated 5'‐ends were self‐ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high‐efficiency transformation. Self‐made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10⁵ cfu/µg of the self‐ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6‐phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5‐fold improved catalytic efficiency (k_cat/K_m) on NAD⁺ as compared to the wild‐type. This protocol is DNA‐sequence independent, and does not require restriction enzymes, special E. coli host, or labor‐intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids.     

Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h

Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.     

Chased PCR: A modified inverse PCR technique to characterize flanking regions of AT-rich DNA fragments

The chased polymerase chain reaction (PCR) technique described here is a convenient method enabling the characterization of flanking regions of a known A/T-rich DNA fragment in two different successive steps. The first step includes a modified inverse PCR with inverted tail-to-tail primers, each with 35 overhanging nucleotides for the insertion into a carrier plasmid. The second step consists of a technique similar to a site-directed mutagenesis. The chased PCR technique is simple, quick, versatile and efficient; it improves the inverse PCR technique and may be applied to any ligation-linker method. Consequently, the techniques for PCR-based gene isolation are more suitable for the isolation of missing sequences of A/T-rich or complex DNA.     

Detection of IncP replicon-specific regions in DNA from Antarctic microbiota

Plasmids capable of horizontal transfer contribute to the adaptability of bacteria, as they may provide genes that enable their hosts to cope with different selective pressures. Only limited information is available on plasmids from Antarctic habitats, and up until now surveys have only used traditional methods of endogenous plasmid isolation. The method based on primer systems, designed on the basis of published sequences for plasmids from different incompatibility (Inc) groups, is appropriate to detect the replicon-specific regions of corresponding plasmids in cultured bacteria, or in total community DNA, which share sufficient DNA similarity with reference plasmids at the amplified regions. In this study, we applied broad-host-range plasmid-specific primers to DNA from microbial samples collected at six different locations in Northern Victoria Land (Antarctica). DNA preparations were used as targets for PCR (polymerase chain reaction) amplification with primers for the IncP (trfA2) and IncQ (oriV ) groups. PCR products were Southern blotted and hybridized with PCR-derived probes for trfA2 and oriV regions. This approach detected the occurrence of IncP-specific sequences in eight out of fifteen DNA samples, suggesting a gene-mobilizing capacity within the original habitats.     

用基因组DNA剪接技术克隆SIgA相关基因

目的：克隆分泌型IgA（SIgA）相关基因--J链基因(IgJ)、多聚免疫球蛋白受体基因（pIgR）和IgA重链恒定区基因（IGHA）,为进一步构建SIgA真核表达质粒奠定基础。方法：采用本室建立的"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果：PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明本实验获得的上述基因与GenBank中的目标基因序列完全一致。结论：本文通过基因组DNA剪接技术成功克隆人类SIgA三个相关基因,提示此技术是合成多外显子cDNA的有效手段。     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

鼠伤寒沙门菌spvBC基因编辑株的构建

利用λRed重组系统和pBAD原核表达载体构建鼠伤寒沙门菌spvBC质粒毒力基因修饰菌株,为深入探究沙门菌毒力基因spv的功能和致病机制及宿主抗感染免疫提供工具菌。以pKD4为模板,PCR扩增含spvBC同源臂的卡那霉素抗性基因以构建同源打靶片段,再将其电转入含有质粒pKD46的鼠伤寒沙门菌中进行同源重组,随后将质粒pCP20电转导入阳性转化子,消除卡那霉素抗性基因,PCR鉴定敲除株的构建。PCR扩增含酶切位点的spvBC基因片段,扩增产物与原核表达载体pBAD/gⅢ分别双酶切后连接构建pBAD-spvBC重组质粒,PCR筛选阳性菌落并测序鉴定。将构建成功的pBAD-spvBC重组质粒电转导入spvBC敲除株中,Western blot测定不同浓度L-阿拉伯糖诱导SpvB和SpvC蛋白表达情况。PCR结果表明鼠伤寒沙门菌spvBC基因敲除成功;PCR及测序结果表明pBAD-spvBC重组质粒构建成功,Western blot结果表明13 mmol/L L-阿拉伯糖可诱导SpvB和SpvC蛋白正常表达。λRed重组系统可用于沙门菌质粒上大片段基因的敲除,pBAD原核表达载体可用于沙门菌质粒上大片段基因的回补,丰富了细菌质粒的基因修饰和编辑策略。     

Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content

Bioactive amounts of antibiotics as well as resistant bacteria reach the soil through manure fertilization. We investigated plasmids that may stimulate the environmental spread and interspecies transfer of antibiotic resistance. After treatment of two soils with manure, either with or without the sulfonamide antibiotic sulfadiazine, a significant increase in copies of the sulfonamide resistance gene sul2 was detected by qPCR. All sul2 carrying plasmids, captured in Escherichia coli from soil, belonged to a novel class of self-transferable replicons. Manuring and sulfadiazine significantly increased the abundance of this replicon type in a chemically fertilized but not in an annually manured soil, as determined by qPCR targeting a transfer gene. Restriction patterns and antibiograms showed a considerable diversity within this novel plasmid group. Analysis of three complete plasmid sequences revealed a conserved 30 kbp backbone with only 36% G+C content, comprised of transfer and maintenance genes with moderate homology to plasmid pIPO2 and a replication module ( rep and oriV ) of other descent. The plasmids differed in composition of the 27.0–28.3 kbp accessory region, each of which carried IS CR2 and several resistance genes. Acinetobacter spp. was identified as a potential host of such LowGC-type plasmids in manure and soil.     

The 79,370-bp conjugative plasmid pB4 consists of an IncP-1beta backbone loaded with a chromate resistance transposon,the strA-strB streptomycin resistance gene pair,the oxacillinase gene bla(NPS-1), and a tripartite antibiotic efflux system of the resistance-nodulation-division family

Plasmid pB4 is a conjugative antibiotic resistance plasmid, originally isolated from a microbial community growing in activated sludge, by means of an exogenous isolation method with Pseudomonas sp. B13 as recipient. We have determined the complete nucleotide sequence of pB4. The plasmid is 79,370 bp long and contains at least 81 complete coding regions. A suite of coding regions predicted to be involved in plasmid replication, plasmid maintenance, and conjugative transfer revealed significant similarity to the IncP-1beta backbone of R751. Four resistance gene regions comprising mobile genetic elements are inserted in the IncP-1beta backbone of pB4. The modular 'gene load' of pB4 includes (1) the novel transposon Tn 5719 containing genes characteristic of chromate resistance determinants, (2) the transposon Tn 5393c carrying the widespread streptomycin resistance gene pair strA-strB, (3) the beta-lactam antibiotic resistance gene bla(NPS-1) flanked by highly conserved sequences characteristic of integrons, and (4) a tripartite antibiotic resistance determinant comprising an efflux protein of the resistance-nodulation-division (RND) family, a periplasmic membrane fusion protein (MFP), and an outer membrane factor (OMF). The components of the RND-MFP-OMF efflux system showed the highest similarity to the products of the mexCD-oprJ determinant from the Pseudomonas aeruginosa chromosome. Functional analysis of the cloned resistance region from pB4 in Pseudomonas sp. B13 indicated that the RND-MFP-OMF efflux system conferred high-level resistance to erythromycin and roxithromycin resistance on the host strain. This is the first example of an RND-MFP-OMF-type antibiotic resistance determinant to be found in a plasmid genome. The global genetic organization of pB4 implies that its gene load might be disseminated between bacteria in different habitats by the combined action of the conjugation apparatus and the mobility of its component elements.