Harnessing the potential of microalgae for the production of monoclonal antibodies and other recombinant proteins.

Monoclonal antibodies (mAbs) have become indispensable tools in various fields, from research to therapeutics, diagnostics, and industries. However, their production, primarily in mammalian cell culture systems, is cost-intensive and resource-demanding. Microalgae, diverse photosynthetic microorganisms, are gaining attention as a favorable option for manufacturing mAbs and various other recombinant proteins. This review explores the potential of microalgae as a robust expression system for biomanufacturing high-value proteins. It also highlights the diversity of microalgae species suitable for recombinant protein. Nuclear and chloroplast genomes of some microalgae have been engineered to express mAbs and other valuable proteins. Codon optimization, vector construction, and other genetic engineering techniques have significantly improved recombinant protein expression in microalgae. These accomplishments demonstrate the potential of microalgae for biopharmaceutical manufacturing. Microalgal biotechnology holds promise for revolutionizing the production of mAbs and other therapeutic proteins, offering a sustainable and cost-effective solution to address critical healthcare needs.

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study.

Angiotensin-converting enzyme 2 (ACE2) is the receptor that enables SARS-CoV-2 to invade host cells. Previous studies have reported that reducing ACE2 expression may have an anti-SARS-CoV-2 effect. In this study, we constructed a pGL4.10-F2-ACE2 vector with double luciferase genes (firefly and Renilla luciferase) under the control of the ACE2 promoter and used it to screen compounds from Chinese traditional medicinal herbs (CTMHs) that can inhibit ACE2 transcription in human cells. We transfected HEK293T cells with pGL4.10-F2-ACE2 and treated them with CTMH compounds and then measured fluorescence to evaluate the indirect inhibition of ACE2 transcription. Out of 37 compounds tested, andrographolide demonstrated a dose-dependent inhibition of ACE2 transcription. We further confirmed by RT-qPCR and Western blot assays that andrographolide also reduced ACE2 expression in BEAS-2B cells in a dose-dependent manner. Moreover, pseudovirus infection assays in BEAS-2B cells demonstrated that andrographolide can inhibit SARS-CoV-2 infection in a dose-dependent manner. These results suggest that andrographolide has potential anti-SARS-CoV-2 activity and could be a candidate drug for COVID-19 prevention and treatment.

The NAC Transcription Factor PgNAC41-2 Gene Involved in the Regulation of Ginsenoside Biosynthesis in Panax ginseng.

Ginseng (Panax ginseng C.A. Meyer) is a perennial herb of the Araliaceae family, a traditional and valuable Chinese herb in China. The main active component of ginseng is ginsenoside. The NAC transcription factors belong to a large family of plant-specific transcription factors, which are involved in growth and development, stress response and secondary metabolism. In this study, we mapped the NAC gene family on 24 pairs of ginseng chromosomes and found numerous gene replications in the genome. The NAC gene PgNAC41-2, found to be highly related to ginsenoside synthesis, was specifically screened. The phylogeny and expression pattern of the PgNAC41-2 gene were analyzed, along with the derived protein sequence, and a structure model was generated. Furthermore, the PgNAC41-2 gene was cloned and overexpressed by a Rhizobium rhizogenes mediated method, using ginseng petioles as receptor material. The saponin content of the transformed material was analyzed to verify the function of the NAC transcription factor in ginseng. Our results indicate that the PgNAC41-2 gene positively regulates the biosynthesis of saponins.

Guidelines on Designing MicroRNA Sponges: From Construction to Stable Cell Line.

Single microRNA (miRNA) can be inhibited using antagomiR which efficiently knocks down a specific miRNA. However, the effect is transient and often results in subtle phenotype. Here we report a guideline on designing miRNA sponges inhibiting a miRNA family. As a model system, we targeted miR-30 family, known as tumor suppressor miRNAs in multiple tumors. To achieve an efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS. The protocol here demonstrates the miRNA sponge as a useful tool to examine the functional impact of inhibition miRNAs.

Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines.

Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C. tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb.

[Effect of miR-204 targeted regulation of DVL3 gene in silica-induced mouse lung epithelial cells].

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.

A novel system for the generation of baculoviruses mutant for an essential gene.

BACKGROUND: Currently, to delete an essential gene from a baculovirus genome, a cell line stably expressing the gene to be knocked-out should be first generated, which is time-consuming. Alternatively, essential genes can be deleted in E. coli using the λ Red recombination system, which requires an electroporation system. Here, based on homologous recombination in insect cells, we develop an alternative efficient system that requires neither generation of a cell line nor an electroporation system. METHODS AND RESULTS: Using puc19-based inverse PCR, a transfer vector for deleting BmNPV orf92 (Bm92, an essential gene) was efficiently constructed. A copy of Bm92 was introduced into the polyhedrin locus of BmNPV bacmid. The transfer vector was then co-transfected into BmN cell with the modified bacmid to enable homologous recombination at the Bm92 locus. An agarose-free approach was developed for the purification of Bm92-disrupted bacmid viruses in insect cells. Subsequently, BmN cells were co-infected with purified Bm92-disrupted bacmid viruses and unmodified bacmid viruses to allow recombination at the Tn7 insertion site between the two viruses. Finally, bacmid DNA extracted from BmN cells was transformed into chemically-treated competent DH10B cells, and blue colonies containing Bm92-disrupted bacmid were selected using PCR. CONCLUSIONS: For its efficiency and convenience, the system has great potential to be used for the generation of baculovirus knockout mutants.

Characterization of neutralizing human anti-tetanus monoclonal antibodies produced by stable cell lines

Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C.tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb.

Bioinformatics analysis on structure and function and expression vector construction of SARS-CoV-2 related protein TMPRSS2 / 中国药理学通报

Aim Human TMPRSS2 is a transmembrane serine protease.In this paper, the structure and func¬tion of the protein were systematically analyzed by bioinformatics, the codon was optimized and the pro- karvotie expression vector was constructed to explore the molecular mechanism of SARS-CoV-2 infecting host cells.Methods The recombinant expression vector pET-22b-TMPRSS2 was generated by molecular clo¬ning technology.The homology, functional sites, sub¬cellular localization, three-dimensional structure and evolutionary characteristics of TMPRSS2 protein were systematically analyzed by using analytical tools such as Protparam, NetPhos3.1, Blast, Clustal X2 and MEGA7.0.Results The prokarvotic expression plas- mid was constructed correctly; TMPRSS2 belongs to medium molecular weight protein, which is composed of 492 amino acid residues.The theoretical isoelectric point is 8.12, the molecular extinction coefficient is 118 145 L • mol~1 • cm"1 , and the half-life is 30 h; TMPRSS2 has 15 potential glycosylation sites and 49 possible phosphorylation sites.It is a transmembrane hydrophilie protein without signal sequenee.In addi¬tion, the protein has 13 potential B-cell epitopes and 7 T-eell epitopes.Seeondarv structure analysis showed that random coil accounted for the highest proportion of TMPRSS2 protein ( 0.453 3) , followed by extended strand (0.252 0).Sequence comparison and evolu¬tionary analysis showed that the highest sequence con¬sistency and closest genetic relationship with human TMPRSS2 was Pan troglodytes, followed by gorilla.Conclusions Human-derived TMPRSS2 protein is ev- olutionarilv conserved and functionally important.Hie results of this study can help to reveal the structure and mechanism of action of TMPRSS2 protein, provide ide¬as for the diagnosis and treatment of COYID-19, and accelerate the research and development process of new drugs targeting TMPRSS2 protein.

Effect of miR-204 targeted regulation of DVL3 gene in silica-induced mouse lung epithelial cells / 中华劳动卫生职业病杂志

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.

A Methodological Advance of Tobacco Rattle Virus-Induced Gene Silencing for Functional Genomics in Plants.

As a promising high-throughput reverse genetic tool in plants, virus-induced gene silencing (VIGS) has already begun to fulfill some of this promise in diverse aspects. However, review of the technological advancements about widely used VIGS system, tobacco rattle virus (TRV)-mediated gene silencing, needs timely updates. Hence, this article mainly reviews viral vector construction, inoculation method advances, important influential factors, and summarizes the recent applications in diverse plant species, thus providing a better understanding and advice for functional gene analysis related to crop improvements.

[Expression and function analysis of FaCO gene in Festuca arundinacea].

Photoperiod plays an important role in transformation from vegetative growth to reproductive growth in plants. CONSTANS (CO), as a unique gene in the photoperiod pathway, responds to changes of day length to initiate flowering in the plant. In this study, the expression level of FaCONSTANS (FaCO) gene under long-day, short-day, continuous light and continuous darkness conditions was analyzed by real-time quantitative PCR. We constructed the over-expression vector p1300-FaCO and infected into Arabidopsis thaliana by Agrobacterium-mediated method. We constructed the silencing vector p1300-FaCO-RNAi and infected into Festuca arundinacea by Agrobacterium-mediated method. The expression of FaCO gene was regulated by photoperiod. The over-expression of FaCO promoted flowering in wild type of Arabidopsis thaliana under long day condition and rescued the late flowering phenotype in co-2 mutant of Arabidopsis thaliana. Silencing FaCO gene in Festuca arundinacea by RNAi showed late-flowering phenotype or always kept in the vegetative growth stage. Our understanding the function of FaCO in flowering regulation will help further understand biological function of this gene in Festuca arundinacea.

Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

Expression and function analysis of FaCO gene in Festuca arundinacea / 生物工程学报

Photoperiod plays an important role in transformation from vegetative growth to reproductive growth in plants. CONSTANS (CO), as a unique gene in the photoperiod pathway, responds to changes of day length to initiate flowering in the plant. In this study, the expression level of FaCONSTANS (FaCO) gene under long-day, short-day, continuous light and continuous darkness conditions was analyzed by real-time quantitative PCR. We constructed the over-expression vector p1300-FaCO and infected into Arabidopsis thaliana by Agrobacterium-mediated method. We constructed the silencing vector p1300-FaCO-RNAi and infected into Festuca arundinacea by Agrobacterium-mediated method. The expression of FaCO gene was regulated by photoperiod. The over-expression of FaCO promoted flowering in wild type of Arabidopsis thaliana under long day condition and rescued the late flowering phenotype in co-2 mutant of Arabidopsis thaliana. Silencing FaCO gene in Festuca arundinacea by RNAi showed late-flowering phenotype or always kept in the vegetative growth stage. Our understanding the function of FaCO in flowering regulation will help further understand biological function of this gene in Festuca arundinacea.

Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.

[Prokaryotic expression, purification and functional identification of epidermal pattern factors in Arabidopsis thaliana].

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl ß-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.

Effect of lentiviral vector mediated RNA binding protein quaking-5 on the proliferation and apoptosis of lung adenocarcinoma cell line H1299 / 解剖学报

To investigate the proliferation and apoptosis of lung adenocarcinoma cells line HI299 by the lentiviral vector mediated RNA binding protein quaking-5 ( QKI-5). Methods GV358 ( up-regulation ) and GV248 ( down-regulation) vectors were used to construct the lentiviral QKI-5 up-regulation vector and down-regulation vector, respectively. The vectors were transfected into 293T cells for lentiviral packaging and viral titer were then determined. Gene sequencing was performed to screen the sequence of vectors. Then Real-time PCR was used to evaluate the expression of QKI-5 mRNA and the proliferation of H1299 cells was examined by colony forming assay after transfection. The apoptosis of HI299 cells was determined by the detection of the expression membrane protein V (annexin V ) and propyl iodide ingot (PI) by flow cytometry. Pro-apoptotic protein Caspae-3 and Caspase-8 were evaluated by Western blotting. Results QKI-5 up-regulation and down-regulation lentiviral vectors were constructed successfully. Compared with the controls, the expression of QKI-5 mRNA of HI299 cells was up-regulated, the cell colony formation was decreased, and the early apoptosis of HI299 cells was increased with the over-expression of Caspase-8 after transfected with up-regulated vector, whereas transfecting with QKI-5 down-regulated vector had opposite effect. Conclusion Lenviral vector mediated QKI-5 could inhibit proliferation and promote apoptosis of lung adenocarcinoma cells through Caspase-8.

Prokaryotic expression, purification and functional identification of epidermal pattern factors in Arabidopsis thaliana / 生物工程学报

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.

Nimble Cloning: A Simple, Versatile, and Efficient System for Standardized Molecular Cloning.

Molecular cloning is one of the most fundamental technologies in molecular biology, and has been critical for driving biotechnological advances. In this study, we have developed a novel method for standardized molecular cloning. The cloning technique known as "Nimble Cloning" uses the restriction enzyme, SfiI, in combination with the T5 exonuclease, to linearize the vector and generate 3'-overhangs simultaneously. Both PCR products and plasmids can be used for the cloning reaction in the Nimble Cloning system. The cloning system is highly efficient, suitable for gene expression in both prokaryotic and eukaryotic expression systems, and enables the reuse of DNA fragments or plasmid entry clones. Nimble Cloning is applicable for the cloning of single or multiple fragments, as well as multi-site cloning. Due also to its simplicity and versatility, the cloning method has great potential for the modular assembly of DNA constructs.