Investigation of Bacillus Thuringiensis Plasmid Instability and its Effect on the Synthesis of Crystals

In order to explore plasmid instability in Bt, four Bt strains belonging to two Bt subspecies were cultured at 42°C for 9 days. HD1 and QBT376 belong to subspecies kurstaki, while H14 and QBT218 belong to subspecies israelensis. Results showed 100% crystal loss for H14 and QBT218, while 76% and 90% crystal loss for HD1 and QBT376, respectively, showing that cry-carrying plasmids are more stable in Bt kurs. than in Bt isr.. HD1, QBT376, and QBT218 cured clones showed significant protease activity compared to their non-cured counterparts. Microscopic observation revealed the delay of sporulation for high number of HD1 and QBT376 cry- clones, while the absence of spores in several H14 and QBT218 cry- clones. Spo-cry- clones of Bti strains had irregular elongated cell shape. Kinetics/day of plasmid curing for H14 and QBT218 showed H14 to have higher pBtoxis plasmid stability. The number of vegetative cells in Bti strains increased with the increase of curing period. As an attempt to create hybrid Bt strains, cry1Aa gene was extracted to transform cured and non-cured strains.

Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

SummaryA growing number of CRISPR-Cas9 associated applications require co-expression of two distinct gRNAs. However, coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid instability, due to the presence of direct repeats (DRs). In this study, deletion between DRs occurred with high frequencies during plasmid construction and duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). Using RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that relied on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

Role of Transcription in Plasmid Maintenance in the hpr1Δ Mutant of Saccharomyces cerevisiae

ABSTRACT The Saccharomyces cerevisiae hyperrecombination mutation hpr1Δ results in instability of sequences between direct repeats that is dependent on transcription of the repeat. Here it is shown that the HPR1 gene also functions in plasmid stability in the presence of destabilizing transcription elongation. In the hpr1Δ mutant, plasmid instability results from unchecked transcription elongation, which can be suppressed by a strong transcription terminator. The plasmid system has been used to examine in vivo aspects of transcription in the absence of Hpr1p. Nuclear run-on studies suggest that there is an increased RNA polymerase II density in the hpr1Δ mutant strain, but this is not accompanied by an increase in accumulation of cytoplasmic mRNA. Suppression of plasmid instability in hpr1Δ can also be achieved by high-copy expression of the RNA splicing factor SUB2, which has recently been proposed to function in mRNA export, in addition to its role in pre-mRNA splicing. High-copy-number SUB2 expression is accompanied by an increase in message accumulation from the plasmid, suggesting that the mechanism of suppression by Sub2p involves the formation of mature mRNA. Models for the role of Hpr1p in mature mRNA formation and the cause of plasmid instability in the absence of the Hpr1 protein are discussed.