Basic Molecular Biology Techniques Research Articles

An Uncharacterised lncRNA Coded by the ASAP1 Locus Is Downregulated in Serum of Type 2 Diabetes Mellitus Patients.

Diabetes mellitus (DM) is a complex and multifactorial disease characterised by high blood glucose. Type 2 Diabetes (T2D), the most frequent clinical condition accounting for about 90% of all DM cases worldwide, is a chronic disease with slow development usually affecting middle-aged or elderly individuals. T2D represents a significant problem of public health today because its incidence is constantly growing among both children and adults. It is also estimated that underdiagnosis prevalence would strongly further increase the real incidence of the disease, with about half of T2D patients being undiagnosed. Therefore, it is important to increase diagnosis accuracy. The current interest in RNA molecules (both protein- and non-protein-coding) as potential biomarkers for diagnosis, prognosis, and treatment lies in the ease and low cost of isolation and quantification with basic molecular biology techniques. In the present study, we analysed the transcriptome in serum samples collected from T2D patients and unaffected individuals to identify potential RNA-based biomarkers. Microarray-based profiling and subsequent validation using Real-Time PCR identified an uncharacterised long non-coding RNA (lncRNA) transcribed from the ASAP1 locus as a potential diagnostic biomarker. ROC curve analysis showed that a molecular signature including the lncRNA and the clinicopathological parameters of T2D patients as well as unaffected individuals showed a better diagnostic performance compared with the glycated haemoglobin test (HbA1c). This result suggests that the application of this biomarker in clinical practice would help to improve the diagnosis, and therefore the clinical management, of T2D patients. The proposed biomarker would be useful in the context of predictive, preventive, and personalised medicine (3PM/PPPM).

Three-dimensional human neural culture on a chip recapitulating neuroinflammation and neurodegeneration.

Neuroinflammation has either beneficial or detrimental effects, depending on risk factors and neuron-glia interactions in neurological disorders. However, studying neuroinflammation has been challenging due to the complexity of cell-cell interactions and lack of physio-pathologically relevant neuroinflammatory models. Here, we describe our three-dimensional microfluidic multicellular human neural culture model, referred to as a 'brain-on-a-chip' (BoC). This elucidates neuron-glia interactions in a controlled manner and recapitulates pathological signatures of the major neurological disorders: dementia, brain tumor and brain edema. This platform includes a chemotaxis module offering a week-long, stable chemo-gradient compared with the few hours in other chemotaxis models. Additionally, compared with conventional brain models cultured with mixed phenotypes of microglia, our BoC can separate the disease-associated microglia out of heterogeneous population and allow selective neuro-glial engagement in three dimensions. This provides benefits of interpreting the neuro-glia interactions while revealing that the prominent activation of innate immune cells is the risk factor leading to synaptic impairment and neuronal loss, validated in our BoC models of disorders. This protocol describes how to fabricate and implement our human BoC, manipulate in real time and perform end-point analyses. It takes 2 d to set up the device and cell preparations, 1-9 weeks to develop brain models under disease conditions and 2-3 d to carry out analyses. This protocol requires at least 1 month training for researchers with basic molecular biology techniques. Taken together, our human BoCs serve as reliable and valuable platforms to investigate pathological mechanisms involving neuroinflammation and to assess therapeutic strategies modulating neuroinflammation in neurological disorders.

Use of a Bacterial Artificial Chromosome to Generate Recombinant SARS-CoV-2 Expressing Robust Levels of Reporter Genes.

Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have engineered reporter-expressing recombinant viruses in which we placed the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression, which facilitates the study of SARS-CoV-2 in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the entire SARS-CoV-2 genome (~30,000 nt) is challenging. Herein, we provide the technical details to engineer rSARS-CoV-2 using the BAC-based RG approach. We describe (i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, (ii) verification of pBeloBAC-FL, (iii) cloning of a Venus reporter gene into pBeloBAC-FL, and (iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with knowledge of basic molecular biology and gene engineering techniques will be able to generate wild-type (WT) and reporter-expressing rSARS-CoV-2. IMPORTANCE We have established a bacterial artificial chromosome (BAC)-based RG system to generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) and to engineer reporter-expressing recombinant viruses to assess viral infection in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the full-length SARS-CoV-2 genome of ~30,000 nucleotides is challenging. Here, we provide all the detailed experimental steps required for the successful generation of wild-type (WT) recombinant SARS-CoV-2 (rSARS-CoV-2). Likewise, we provide a comprehensive protocol on how to generate and rescue rSARS-CoV-2 expressing high levels of a Venus fluorescent reporter gene from the locus of the viral nucleocapsid (N) protein. By following these protocols, researchers with basic knowledge in molecular biology will be able to generate WT and Venus-expressing rSARS-CoV-2 within 40 days.

Enzyme-free targeted DNA demethylation using CRISPR-dCas9-based steric hindrance to identify DNA methylation marks causal to altered gene expression.

DNA methylation involves the enzymatic addition of a methyl group primarily to cytosine residues in DNA. This protocol describes how to produce complete and minimally confounded DNA demethylation of specific sites in the genome of cultured cells by clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and without the involvement of an epigenetic-modifying enzyme, the purpose of which is the evaluation of the functional (i.e., gene expression or phenotypic) consequences of DNA demethylation of specific sites that have been previously implicated in particular pathological or physiological contexts. This protocol maximizes the ability of the easily reprogrammable CRISPR-dCas9 system to assess the impact of DNA methylation from a causal rather than correlational perspective: alternative protocols for CRISPR-dCas9-based site-specific DNA methylation or demethylation rely on the recruitment of epigenetic enzymes that exhibit additional nonspecific activities at both the targeted site and throughout the genome, confounding conclusions of causality of DNA methylation. Inhibition or loss of DNA methylation is accomplished by three consecutive lentiviral transductions. The first two lentiviruses establish stable expression of dCas9 and a guide RNA, which will physically obstruct either maintenance or de novo DNA methyltransferase activity at the guide RNA target site. A third lentivirus introduces Cre recombinase to delete the dCas9 transgene, which leads to loss of dCas9 from the target site, allowing transcription factors and/or the transcription machinery to interact with the demethylated target site. This protocol requires 3-8 months to complete owing to prolonged cell passaging times, but there is little hands-on time, and no specific skills beyond basic molecular biology techniques are necessary.

Lab-It Is Taking Molecular Genetics to School

The Molecular Genetics Mobile Lab or “Laboratório itinerante de Genética Molecular” (Lab-it) was funded in 2008 by Leonor Cancela to promote the learning of molecular genetics which had been introduced at that time into high school biology programms. The project aimed to introduce hands-on laboratory activities in molecular genetics to complement the theoretical concepts taught in school. These included the development of experimental protocols based on theoretical scenarios focusing on themes of forensics sciences, biomedical applications, diagnostic methods, and ecological research using basic molecular biology techniques, such as DNA extraction, polymerase chain reaction (PCR), electrophoresis, and restriction enzyme application. In these scenarios, the students execute all the procedures with the help of the Lab-it instructor and using the Lab-it equipment, followed by a discussion of the results with all the participants and the school teacher. These approaches help the students to consolidate the concepts of molecular biology and simultaneously promote discussions on new advances in the area and choices for university careers. In addition to practical sessions, Lab-it also promotes seminars on topics of interest to the students and teachers. Since 2008, 18 high schools have participated in the region of Algarve, averaging each year about 400 students participating in practical activities. In 2021, despite the COVID pandemic, 9 schools and 379 students were involved in Lab-it practical sessions and 99% of them considered the activity to contribute to better understanding the molecular biology methods approached in theoretical classes and expressed high interest in those sessions.

Microbial production of 2,3-butanediol from rice husk using anaerobic Clostridium species

Interest in the area of biomass based-product production is increasing all over the world due to the environmental challenges posed by fossil fuel and fear of its extinction. Production of biofuel and other compounds especially from agricultural waste can reduce these environmental problems because of its sustainability and environmentally friendliness. One of the major petrochemical product widely used in many industries is 2,3-butanediol and was found to be produced from agricultural wastes by microorganisms. Therefore, Microbial production of 2,3-butanediol from rice husk using Clostridium species was investigated in this research. Structural composition of the rice husk was determined before and after pretreatment. Hemicellulose and lignin content of rice husk was determined after extraction while cellulose was determined as the difference from the extractives, hemicelluloses and lignin. Dilute (2%) NaOH was used for the pretreatment of rice husk. Hydrolysis was carried out using Aspergillus niger and reducing sugar released was determined using standard method with UV-VIS spectrophotometer. Clostridium species was isolated from sugarcane bagasse, identified using basic morphological and molecular biology techniques. The fermentation of rice husk was performed using the Clostridium species. Fermentation by-product was determined using Gas Chromatography Mass-spectrometry. Cellulose content increased from 32% before pretreatment to 53.3% after pretreatment, lignin increased from 8.4% before pretreatment to 30.7% after pretreatmemt and hemicellulose decreased from 30% before pretreatment to 8% after pretreatment. A total of 1.05 g/l of reducing sugar was released after enzymatic hydrolysis of the rice husk with Aspergillus niger. Alcohol 2,3-butanediol (0.6%) and Furfuryl alcohol (0.45%) were detected in the by-product of fermentation. Other compounds detected are fatty acids that ranges from C16 to C25 with 9,12-Octadecanoic acid as the major fatty acid. From the results of this work, Rice husk was found to have substantial amount of sugar (cellulose and hemicelluce) that can be converted to valuable product including 2,3-butanediol.
 Keywords: Biofuel, Bio-refinary, Cellulose, Clostrudium, fermentation.

CUREing cancer: Development and implementation of a molecular biology-focused course-based undergraduate research experience using a cancer cell culture model.

Many students in the sciences are interested in exploring research opportunities; however, the one-on-one faculty mentorship model often lacks the ability to supervise large numbers of students. An alternative mechanism for exposing undergraduate students to the research process is participation in a Course-based Undergraduate Research Experience (CURE). CUREs promote inclusivity in research, and provide structure for both students and faculty while engaging students in scientific discovery. This study describes a model for a CURE in cancer biology, and reports student outcomes. Students utilized bioinformatics to predict targets genes of miR-100, a microRNA that is differentially expressed in a cell culture model of breast cancer metastasis. Students were required to engage with primary literature to write a grant proposal for their target gene, and then were trained to perform basic molecular biology techniques to test their individual hypotheses. Additionally, the course integrated opportunities to troubleshoot experiments and present data to the group, and culminated in a publication style scientific report discussing the results of their individual research project. Students reported significantly increased confidence in executing various molecular biology techniques and research-related skills based on pre- and post-assessment surveys. Student feedback also indicated that they gained an understanding of primary literature, experimental design, and scientific writing as a result of the course. This study supports that CUREs can be an effective pedagogy for not only engaging larger groups of students in research, but also improving their confidence and skill set in the laboratory.

Abstract PR06: Dissecting mechanisms of replication fork stabilization in patient-derived high-grade serous organoid cultures and their impact on therapeutic sensitivity and the immune-tumor interaction

Abstract Genomic analyses indicate that 50% of high-grade serous ovarian cancers (HGSC) harbor a genomic alteration in a DNA damage repair gene that may lead to functional defects. Using functional assays on patient-derived HGSC organoid cultures to test the capacity of the tumor cells to repair double-strand DNA breaks and to protect stalled replication forks, we have found that many HGSCs have stalled fork protection defects regardless of the genomic background of the tumor and that these defects correlate with sensitivity to replication stress inducing therapeutic agents. We hypothesized that gaining a better understanding of the mechanisms of replication fork instability and stability in HGSC organoid cultures would help to better understand the mechanisms of therapeutic sensitivity of the tumor cells. The purpose of this work is to understand how replication fork stabilization either in the primary tumor or through selection post-treatment leads to alterations in tumor cell biology, including therapeutic sensitivity and interaction of the tumor cells with the surrounding microenvironment. We utilized bulk RNA sequencing analysis of HGSC organoid cultures with varied replication fork protection capacity, some matched pairs of untreated and post-neoadjuvant tumors, to stratify differences in functional profiles in fork stable versus unstable tumors; we then used basic molecular biology techniques to understand the mechanisms of fork stabilization and how this stabilization affects the therapeutic sensitivity of the cells. We also developed and utilized multiple functional assays to assess the interaction of HGSC organoids of varying fork protection capacity with their immune microenvironment in different drug exposure settings. We identified multiple proteins that through either up- or downregulation lead to stabilization of replication forks in the tumor cells and found that the mechanisms of stabilization can occur at both the level of the replication fork and the overall transcriptional level of the cell and can alter the therapeutic sensitivity of the cells. We have determined that replication fork stability leads to increased mesenchymal characteristics in tumors and to decreased activation of the antitumor immune response within the cultures after treatment with DNA damage repair and immuno-oncologic (IO) agents. Overall, these results indicate that replication fork stabilization in HGSC through multiple different mechanisms can lead to altered interactions of the tumor cells with their microenvironment and altered therapeutic sensitivity. This abstract is also being presented as Poster B10. Citation Format: Sarah J. Hill, Patrick Lizotte, Nikolas Kesten, Neil S. Horowitz, Michael G. Muto, Michael J. Worley, Colleen M. Feltmate, Ross S. Berkowitz, Henry Long, Ursula A. Matulonis, Christopher P. Crum, Myles Brown, Alan D. D'Andrea. Dissecting mechanisms of replication fork stabilization in patient-derived high-grade serous organoid cultures and their impact on therapeutic sensitivity and the immune-tumor interaction [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR06.

Validation of some disease-resistance molecular markers associated with multiple diseases in tomato for marker-assisted selection program

Marker-assisted selection (MAS) is a tool that is widely applied in tomato resistance breeding. To determine the robustness of some molecular markers commonly used in MAS, extensive screening of 964 tomato lines was performed under a controlled experimental condition. Initially, the application of 36 molecular markers targeting 26 resistance genes (R genes) and 14 major diseases was evaluated. Here, we employed basic molecular biology and bioinformatics techniques for analysis where polymorphism, accuracy and clearness of amplicons constituted the selection criteria of markers. Upon initial analysis, 20 of these markers designated as efficient markers, among which 8 were considered gene-based markers and referred to as perfect markers were selected for detail evaluation. Information extrapolated from PCR result revealed 18 R genes that control 12 diseases were grouped under efficient markers. On the other hand, grouping of breeding lines based on the number of R gene harbored comprehensively revealed 62% of the lines to be void of R gene, while 38% carry different types of R genes. This provides us with an avenue to better understand new sources of resistance in the breeding lines. Conclusively, these efficient markers and their limited PCR condition can be suggested as basis of a diagnostic kit for MAS applications against 12 major tomato diseases and the identified resistant breeding lines could be conserved in order to be propagated as different sources of resistance for the development of new resistant varieties. Therefore, in areas with high vulnerability to diseases, high efficiency combination of the relevant R genes and their pyramiding into commercial tomato varieties are proposed to be implemented as a pragmatic approach.

An idea to explore: Genotyping bull sperm to introduce basic molecular biology techniques in an animal science course.

The laboratory exercise described here aims to provide a relevant context for learning basic DNA techniques in an introductory animal science course at tertiary level. In two 4-hr laboratory sessions, students assess the suitability of bulls for inclusion in a gene-assisted selection program for A2 β-casein by genotyping commercial bull sperm. Sperm cells are lysed to extract the genomic DNA, and PCR with primers for the β-casein gene is performed. Using the principle of amplification-created restriction sites, restriction digestion with TaqI can be used to distinguish between the A1 allele and the A2 allele of the gene. Cut PCR amplicons are separated by gel electrophoresis to evaluate the genotype of each bull. Students then write a diagnostic report with accompanying letter to their fictional client, explaining the DNA test, and interpreting the results. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):708-711, 2019.

Circulating MicroRNAs in Cancer: Potential and Challenge.

MicroRNAs (miRNAs) are endogenous non-coding small RNA molecules that can be secreted into the circulation and exist in remarkably stable forms. Like intercellular miRNAs, circulating miRNAs participate in numerous regulations of biological process and expressed aberrantly under abnormal or pathological status. The quality and quantity changes of circulating miRNAs are associated with the initiation and progression of cancer and can be easily detected by basic molecular biology techniques. Consequently, considerable effort has been devoted to identify suitable extracellular miRNAs for noninvasive biomarkers in cancer. However, several challenges need to be overcome before the practical application. In this review, we discuss several issues of circulating miRNAs: biological function and basic transport carriers; extracellular cell communication process; roles as reliable cancer biomarkers and usage in targeted cancer therapy; and challenges for clinical application.

Undergraduate lab series using the K562 human leukemia cell line: Model for cell growth, death, and differentiation in an advanced cell biology course.

This sequence of labs was developed for an upper level undergraduate cell biology course at Fairfield University. The labs are based on the use of the K562 human erythroleukemia cell line, a model system that is exceptionally amenable to an undergraduate cell biology lab course due to its ease of maintenance and propagation and usefulness for studies of growth, death, and differentiation. The sequence of labs is conducted over a 6-week period, following a series of weekly cell biology labs covering basic cell and molecular biology techniques. Together, the lab series has four primary objectives 1) to teach students how to culture and maintain mammalian cells; 2) to build student competency in standard cell biology techniques; 3) to demonstrate the role of growth factors on cell proliferation and viability; and 4) to provide students with an opportunity to use these cells in an independent investigation on cell differentiation. We provide examples of student data and offer a range of experimental measurements depending on institutional capacity and facilities. Our assessment data suggest that students find great value in this lab series, enhancing their comprehension of key concepts, acquisition of important lab skills, and depth of understanding of the research process. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):263-271, 2019.

Isolation and Characterization of Bacterial Cellulase Producers for Biomass Deconstruction: A Microbiology Laboratory Course.

The conversion of biomass to biofuels presents a solution to one of the largest global challenges of our era, climate change. A critical part of this pipeline is the process of breaking down cellulosic sugars from plant matter to be used by microbes containing biosynthetic pathways that produce biofuels or bioproducts. In this inquiry-based course, students complete a research project that isolates cellulase-producing bacteria from samples collected from the environment. After obtaining isolates, the students characterize the production of cellulases. Students then amplify and sequence the 16S rRNA genes of confirmed cellulase producers and use bioinformatic methods to identify the bacterial isolates. Throughout the course, students learn about the process of generating biofuels and bioproducts through the deconstruction of cellulosic biomass to form monosaccharides from the biopolymers in plant matter. The program relies heavily on active learning and enables students to connect microbiology with issues of sustainability. In addition, it provides exposure to basic microbiology, molecular biology, and biotechnology laboratory techniques and concepts. The described activity was initially developed for the Introductory College Level Experience in Microbiology (iCLEM) program, a research-based immersive laboratory course at the US Department of Energy Joint BioEnergy Institute. Originally designed as an accelerated program for high-potential, low-income, high school students (11th–12th grade), this curriculum could also be implemented for undergraduate coursework in a research-intensive laboratory course at a two- or four-year college or university.

Activation of Split RNA Aptamers: Experiments Demonstrating the Enzymatic Synthesis of Short RNAs and Their Assembly As Observed by Fluorescent Response

With the advancement of ribonucleic acid (RNA) research in the field of biochemistry, ensuring that undergraduate students have both the theoretical and practical knowledge of current, cutting-edge experimental techniques is of marked importance. Many current biochemistry experiments demonstrate various basic molecular biological techniques including isolation and quantification of nucleic acids, protein gel electrophoresis, and DNA amplification reactions. While covering a wide base of skills, there is an underutilization of RNA as a pedagogical tool. This multistep laboratory experiment introduces students to the emerging field of RNA nanotechnology and to the RNA assembly and subsequent conditional activations of preprogrammed functionalities such as fluorescence. To achieve this, a sequence of standard molecular techniques such as PCR, DNA purification, RNA transcription and purification, RNA quantification, and self-assembly followed by analysis with electrophoretic mobility shift assays and spectros...

Generation of Fabs-in-tandem immunoglobulin molecules for dual-specific targeting

Bispecific antibody (BsAb) has become an important trend in developing next generation biologics therapies. By simultaneously engaging two molecular targets, BsAbs show distinctive mechanism of actions that could lead to clinical benefits unattainable by conventional monoclonal antibodies (mAbs). Successful launch provided clinical validation and encourage more BsAb development in the pipeline of pharmaceutical companies. Fabs-in-tandem immunoglobulin (FIT-Ig™) format was initially described in 2017. This unique design provides a symmetrical and tetravalent IgG-like bispecific molecule with correct association of 2 sets of VH/VL pairs, where two Fabs are fused directly in a crisscross orientation without any mutations or use of peptide linkers. FIT-Ig can be readily made from 2 existing monoclonal antibodies by basic molecular biology techniques with high expression level in mammalian cells, and easily purified to homogeneity using standard approaches without extensive optimization. FIT-Ig molecules exhibit favorable drug-like properties, in vitro and in vivo functions, as well as manufacturing efficiency for commercial development. Here, we provide an example of construction and preliminary characterization of a FIT-Ig molecule with discussions on optimization and general utility.

Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

Assessment of knowledge of participants on basic molecular biology techniques after 5‐day intensive molecular biology training workshops in Nigeria

The deployment of molecular biology techniques for diagnosis and research in Nigeria is faced with a number of challenges, including the cost of equipment and reagents coupled with the dearth of personnel skilled in the procedures and handling of equipment. Short molecular biology training workshops were conducted at the Nigerian Institute of Medical Research (NIMR), to improve the knowledge and skills of laboratory personnel and academics in health, research, and educational facilities. Five-day molecular biology workshops were conducted annually between 2011 and 2014, with participants drawn from health, research facilities, and the academia. The courses consisted of theoretical and practical sessions. The impact of the workshops on knowledge and skill acquisition was evaluated by pre- and post-tests which consisted of 25 multiple choice and other questions. Sixty-five participants took part in the workshops. The mean knowledge of molecular biology as evaluated by the pre- and post-test assessments were 8.4 (95% CI 7.6-9.1) and 13.0 (95 CI 11.9-14.1), respectively. The mean post-test score was significantly greater than the mean pre-test score (p < 0.0001). The five-day molecular biology workshop significantly increased the knowledge and skills of participants in molecular biology techniques. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(4):313-317, 2017.

A Modular Approach to Building Complex Synthetic Circuits.

Efficient assembly of genetic circuits is a critical element in synthetic biology for implementing complex gene expression systems. Here we present an assembly framework that integrates recent developments in DNA recombination technologies and unique nucleotide sequences for modular and reliable construction of complex genetic circuits. All relevant protocols are described in detail. Implementing these protocols relies on readily available reagents and basic molecular biology techniques without the need for specialized equipment.

A Do-it-Yourself Protocol for Making Desired Mutant Proteins

The generation of a mutant protein is often crucial to the elucidation of the pathogenesis of a genetic disease. This requires site-directed mutagenesis of an expression vector containing the gene of interest. Current site-directed mutagenesis procedures are derivatives of either a restriction site elimination method or a PCR-based mutagenesis method, which involve a commercial kit manufactured by any one of a number of biotechnology companies. A new efficient economical site-directed mutagenesis procedure is described here to provide researchers an additional choice that does not to rely on a commercial kit. This novel method has been validated, and requires only basic molecular biological techniques and reagents available in most laboratories even in developing countries. The mutation efficiency of this new method is usually greater than 80%. In case several different mutants of a protein are required for elucidation of the protein structure-function relationships, this method has distinct advantages since multiple mutations can be derived from the initial single strand DNA template.

Genome Editing in Human Cells Using CRISPR/Cas Nucleases.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.