A Simplified Gibson Assembly Method for Site Directed Mutagenesis Using Non-Gibson Primers

Background: Site-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired. Method: We present a versatile and simple method to efficiently introduce a variety of mutation schemes using the Gibson-assembly without the need for unique Gibson primers. The method entails use of standard SDM primers (shorter and completely overlapping in sequences in contrast to Gibson primers) that are separately employed with common primer (~25 bps long) for amplification of fragments flanking the site of mutagenesis, followed by rapid amplification of the Gibson-assembled product for added visualization and sequencing steps for ensuring high success rates.Results: We find that assembly of the fragments via the Gibson reaction mixture is attainable within as short as 15 minutes, despite the need for extensive digestion of the DNA (by exonuclease) past the entire SDM primer sequence (to expose non-clashing overlap between the fragments). We also find that the amount of the assembled Gibson product is too low to be visualized and assessed on standard agarose gel. We thereby introduce a short amplification step (by use of the same short primers initially employed) to 1) easily resolve whether the product (only the correct size can yield a product) has been obtained, and 2) for isolation of product for DNA-sequencing (to assess whether mutation(s) have been introduced). No other SDM method enables assessment of mutagenesis prior completion of the process. Conclusion: We employ our approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically in DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). The entire protocol spans only four days, requires minimal primers sets (as well as can be used with most in-house primers) and provides very high yields and success rates (>98%).

Simple and Rapid Assembly of TALE Modules Based on the Degeneracy of the Codons and Trimer Repeats

Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.

A Language for Modeling and Optimizing Experimental Biological Protocols

Automation is becoming ubiquitous in all laboratory activities, moving towards precisely defined and codified laboratory protocols. However, the integration between laboratory protocols and mathematical models is still lacking. Models describe physical processes, while protocols define the steps carried out during an experiment: neither cover the domain of the other, although they both attempt to characterize the same phenomena. We should ideally start from an integrated description of both the model and the steps carried out to test it, to concurrently analyze uncertainties in model parameters, equipment tolerances, and data collection. To this end, we present a language to model and optimize experimental biochemical protocols that facilitates such an integrated description, and that can be combined with experimental data. We provide probabilistic semantics for our language in terms of Gaussian processes (GPs) based on the linear noise approximation (LNA) that formally characterizes the uncertainties in the data collection, the underlying model, and the protocol operations. In a set of case studies, we illustrate how the resulting framework allows for automated analysis and optimization of experimental protocols, including Gibson assembly protocols.

Complete genome sequence and construction of an infectious full-length cDNA clone of a cucumber vein yellowing virus (CVYV) isolate from Portugal

Cucumber vein yellowing virus (CVYV) is a member of the genus Ipomovirus in the family Potyviridae. In the National Center for Biotechnology Information (NCBI) database, three complete genome sequences of CVYV isolates from Spain (NC_006941), Israel (KT276369), and Jordan (JF460793) are available. In this study, we report the complete sequence of an isolate of CVYV from Portugal (DSMZ PV-0776) along with the construction of an infectious full-length cDNA clone via Gibson assembly. The sequence of CVYV Portugal shows the closest relationship to a CVYV isolate from Spain (genome, 99.7% identity; polyprotein, 99.7% identity). The CVYV full-length cDNA clone was introduced by electroporation into Rhizobium radiobacter and infiltrated into the cotyledons of Cucumis sativus plantlets, resulting in symptoms resembling those of the wild-type virus. Transmission of the infectious CVYV full-length clone by the whitefly Bemisia tabaci was confirmed. This first report confirming the infectivity of a CVYV cDNA clone provides the opportunity to study gene functions in a consistent genomic background.

Molekularbiologische Methoden 2.0

In den vergangenen Jahren hat sich die Molekularbiologie rasant weiterentwickelt. Technologien wie die Modulare Klonierung, PCR-basierte Klonierungen und das Gibson Assembly sind inzwischen in vielen Laboren als Standard etabliert. Neben den modernen Klonierungsverfahren wurden die die Denkansätze in der modernen Biologie revolutionierende Bioinformatik und die synthetische Biologie aufgenommen. Die zunehmende Bedeutung reicht inzwischen weit in die Gesellschaft hinein, wie die „Omics“-Technologien und die Genom Editierung zeigen, die ebenfalls in diesem Buch behandelt werden. Die 3. Auflage wurde korrigiert und aktualisiert und enthält viele hilfreiche Tipps und Tricks, welche die Fehlersuche im Labor erleichtern können. Concept-Maps visualisieren Zusammenhänge zwischen den vorgestellten Methoden. Zahlreiche Abbildungen und Tabellen veranschaulichen komplexe Sachverhalte, „Gut zu wissen“-Boxen liefern Hintergrundinformationen, „Tipp“-Boxen geben wertvolle Hinweise für die praktische Arbeit und Protokolle besonders wichtiger Verfahren erleichtern das Verständnis. Ideal für Studium und Praxis!

UNIGEMS: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

Synthetic biology is as an excellent vehicle for education, as it enables creativecombination of engineering and molecular biology approaches for quantitative charac-terisations of the assembled constructs. However, there is a limited number of resourcesavailable for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. To expandthe availability of education-friendly resources to teach synthetic biology and geneticengineering, we developed Unigems, a set of 10 plasmids that enable out-of-the-boxinvestigations of principles of gene expression control, as well as more complex designs- a biological logic gate. The system uses a common high-copy plasmid backbone anda common set of primers to enable Gibson-assembly of PCR-generated or synthesisedparts into a target vector. It currently has two reporter genes with either two consti-tutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as wellas a single-plasmid implementation of an AND logic gate. The Unigems system hasalready been employed in undergraduate teaching settings, during outreach events andfor training of iGEM teams. All plasmids have been deposited in Addgene.

IS26 cannot move alone

Background IS26 plays a major role in the dissemination of antibiotic resistance determinants in Gram-negative bacteria. Objectives To determine whether insertion sequence IS26 is able to move alone (simple transposition) or if it exclusively forms cointegrates. Methods A two-step PCR using outward-facing primers was used to search for circular IS26 molecules. Gibson assembly was used to clone a synthetic IS26 containing a catA1 chloramphenicol resistance gene downstream of the tnp26 transposase gene into pUC19. IS activity in a recA− Escherichia coli containing the non-conjugative pUC19-derived IS26::catA1 construct and the conjugative plasmid R388 was detected using a standard mating-out assay. Transconjugants were screened for resistance. Results Circular IS26 molecules that would form with a copy-out route were not detected by PCR. The synthetic IS26::catA1 construct formed CmRTpR transconjugants (where CmR and TpR stand for chloramphenicol resistant and trimethoprim resistant, respectively), representing an R388 derivative carrying the catA1 gene at a frequency of 5.6 × 10−7 CmRTpR transconjugants per TpR transconjugant, which is comparable to the copy-in activity of the unaltered IS26. To test for simple transposition of IS26::catA1 (without the plasmid backbone), 1200 CmRTpR colonies were screened and all were resistant to ampicillin, indicating that the pUC19 backbone was present. Hence, IS26::catA1 had only formed cointegrates. Conclusions IS26 is unable to move alone and cointegrates are the exclusive end products of the reactions mediated by the IS26 transposase Tnp26. Consequently, when describing the formation of complex resistance regions, simple ‘transposition’ of a single IS26 should not be invoked.

Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by Agrobacterium-mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.