Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylSZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNApyl, such as pylSZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated.

Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon

A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylSZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNApyl, such as pylSZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated.

A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection

The use of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) for sequence-specific elimination of bacteria or resistance genes is a powerful tool for combating antibiotic resistance. However, this approach requires efficient delivery of CRISPR/Cas DNA cassette(s) into the targeted bacterial population. Compared to phage transduction, plasmid conjugation can deliver DNA to a broader host range but often suffers from low delivery efficiency. Here, we developed multi-plasmid conjugation systems for efficient CRISPR/Cas delivery, target DNA elimination and plasmid replacement. The CRISPR/Cas system, delivered via a broad-host-range R1162 mobilizable plasmid, specifically eliminated the targeted plasmid in recipient cells. A self-transmissible RK2 helper plasmid facilitated the spread of mobilizable CRISPR/Cas. The replacement of the target plasmid with another plasmid from the same compatibility group helped speed up target plasmid elimination especially when the target plasmid was also mobilizable. Together, we showed that up to 100% of target plasmid from the entire recipient population could be replaced even at a low (1:180) donor-to-recipient ratio and in the absence of transconjugant selection. Such an ability to modify genetic content of microbiota efficiently in the absence of selection will be critical for future development of CRISPR antimicrobials as well as genetic tools for in situ microbiome engineering.

Plasmid hypermutation using a targeted artificial DNA replisome

Extensive exploration of a protein’s sequence space for improved or new molecular functions requires in vivo evolution with large populations. But disentangling the evolution of a target protein from the rest of the proteome is challenging. Here, we designed a protein complex of a targeted artificial DNA replisome (TADR) that operates in live cells to processively replicate one strand of a plasmid with errors. It enhanced mutation rates of the target plasmid up to 2.3 × 105–fold with only a 78-fold increase in off-target mutagenesis. It was used to evolve itself to increase error rate and increase the efficiency of an efflux pump while simultaneously expanding the substrate repertoire. TADR enables multiple simultaneous substitutions to discover functions inaccessible by accumulating single substitutions, affording potential for solving hard problems in molecular evolution and developing biologic drugs and industrial catalysts.

Reconstitution and visualization of HIV-1 capsid-dependent replication and integration in vitro

During the first half of the viral life cycle, HIV-1 reverse transcribes its RNA genome and integrates the double-stranded DNA copy into a host cell chromosome. Despite progress in characterizing and inhibiting these processes, in situ mechanistic and structural studies remain challenging. This is because these operations are executed by individual viral preintegration complexes deep within cells. We therefore reconstituted and imaged the early stages of HIV-1 replication in a cell-free system. HIV-1 cores released from permeabilized virions supported efficient, capsid-dependent endogenous reverse transcription to produce double-stranded DNA genomes, which sometimes looped out from ruptured capsid walls. Concerted integration of both viral DNA ends into a target plasmid then proceeded in a cell extract–dependent reaction. This reconstituted system uncovers the role of the capsid in templating replication.

Construction of Repulsive Guidance Molecule A-Specific RNA Interference Recombinant Adenovirus

This study aims to construct an RGMa RNAi plasmid in preparation for a subsequent research, and three shRNAs were designed for collection. The designed shRNAs were taken into the target plasmid of pegensil-1 to silence the mRNA of RGMa. Sequencing and enzyme identification were performed, and PC12 cells were transfected with a shRNA-containing plasmid. Then, the effect of the silencing of the gene was tested by RT-PCR using PC12 cells and to choose the best one. The results showed that the first one was choose for the subsequent research among these three designed shRNAs, which had the highest transfection efficiency and a concentration of 2.5 × 109 pfu/ml. The concentration of Adv(–) was 2.1 × 109 pfu/ml for the blank control. The effect of the constructed RGMa RNAi plasmid for the subsequent research is stable and effective.

PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3′ overhangs. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution.