DNA methylases for site-selective inhibition of type IIS restriction enzyme activity.

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques. KEY POINTS: • Non-switchable methylases always inhibit the relevant type IIS endonuclease activity • Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site • Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assembly.

The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

Coexpression and Reconstitution of Enzymatic Cascades in Bacteria Using UbiGate.

Coexpression of multiple genes of interest (GOIs) is advantageous for many purposes including the elucidation of protein complexes, reconstitution of enzymatic cascades that mediate the biosynthesis of compounds, the study of signaling cascades, or the elucidation of posttranslational modification. Additional advantages of coexpressing proteins is increased solubility and stability of proteins. For this purpose we developed UbiGate, a modular system based on Golden Gate cloning that enables the generation of polycistronic expression cassettes. Their generation is achieved in four simple steps: (1) GOIs are amplified via PCR, (2) and restriction-ligated into level 0 cloning vectors. Next, (3) the GOIs in a level 0 vector are restriction-ligated into a dedicated set of level 1 vectors that define the position of the GOI within the operon. In the last step (4), level 1 vectors are cloned into a modified pET28-GG expression vector. The resulting modules at each step can be reused to generate fusions with different tags in any desired order and orientation, to include up to six different proteins representing a useful tool facilitating the study of plant metabolic and signaling pathways.

Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids.

Multiplex hextuple luciferase assaying allows monitoring the activity of five experimental pathways against one control at the same time. To perform multiplex hextuple luciferase assaying, six orthogonal luciferase reporter units are needed of which five are pathway-specific and one acts as a control for normalization. To ensure stoichiometric delivery of all six luciferase reporters in every transfected cell, synthetic assembly DNA cloning is used to stitch together all six luciferase reporter units into a single vector. Here, we provide a detailed three-step synthetic assembly DNA protocol to generate multiplex hextuple luciferase reporter plasmids for any five cellular signaling pathways of interest, against a control normalization pathway. A first protocol is provided on how to generate plasmids that contain novel transcription factor-binding motifs for specific transcription factors. A second protocol details on how to couple these novel transcription factor-binding motifs to one of five orthogonal luciferases to obtain specific luciferase reporters for cellular signaling pathways acting upstream of those transcription factor-binding motifs. Finally, a third protocol provides details on how to assemble orthogonal luciferase reporters for five cellular signaling pathways acting upstream of five unique transcription factor-binding motifs together with a control constitutive pathway luciferase reporter that will be used for normalization to obtain a final multiplex hextuple luciferase vector.

A convenient method to generate and maintain poly(A)-encoding DNA sequences required for in vitro transcription of mRNA.

Generating mRNA in vitro to encode therapeutic or cell-modifying proteins is rapidly gaining favor. An important factor that determines efficiency of translation from in vitro transcribed mRNA is the length of the 3' poly(A) sequence. However, reproducibly generating and maintaining templates from circular plasmids to have consistent lengths of the homo poly(A) sequences is challenging. The procedure reported here entails repeated restriction digestion with type IIS enzymes, ligation and circular plasmid propagation. The homopolymeric sequence of approximately 100 bp that is generated using the method is approximately equal to the number of 3' A residues found in the mRNA of  mammalian cells. Evaluating expression in vivo of a reporter transcript produced using this method showed efficient expression in vivo.