Prospects of CRISPR/Cas cloning vectors to combat antibiotic resistance gene dissemination

CRISPR (Clustered Regularly Interspaced Small Palindromic Repeats) and Cas (CRISPR associated) proteins comprise an adaptive immune system in prokaryotes CRISPR Cas systems digest invading nucleic acids and incorporate a 30 bp snippet (“spacer") of the invader into the host genome Spacers are transcribed into CRISPR RNAs (crRNA's) that recognize the same nucleic acid in the event of a repeat invasion and guide nucleases to the target for elimination We hypothesize that a plasmid vector, when transformed into a host cell, could exogenously express a CRISPR/Cas system and prevent the host’s participation in conjugative processes

Prospects of CRISPR/Cas cloning vectors to combat antibiotic resistance gene dissemination

CRISPR (Clustered Regularly Interspaced Small Palindromic Repeats) and Cas (CRISPR associated) proteins comprise an adaptive immune system in prokaryotes CRISPR Cas systems digest invading nucleic acids and incorporate a 30 bp snippet (“spacer") of the invader into the host genome Spacers are transcribed into CRISPR RNAs (crRNA's) that recognize the same nucleic acid in the event of a repeat invasion and guide nucleases to the target for elimination We hypothesize that a plasmid vector, when transformed into a host cell, could exogenously express a CRISPR/Cas system and prevent the host’s participation in conjugative processes

New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria

New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivo assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium.

Expression of synthetic Phytochelatin EC20 in E. Coli increases its biosorption capacity and cadmium resistance

In this study E. coli recombinant clones that express the EC20 synthetic phytochelatin intracellularly were constructed. The increasement of Cd2+ biosorption capacity, and, also, the increasement of resistance to this toxic metal were analyzed. A gene that encodes the synthetic phytochelatin EC20 was synthesized in vitro. The EC20 synthetic gene was amplified by PCR, inserted into the DNA cloning vectors pBluescript®KS+ and pGEM®-TEasy, and also into the expression vectors pTE [pET-28(a)® derivative] and pGEX-T4-2®. The obtained recombinant plasmids were employed for genetic transformation of E. coli: pBsKS-EC20 and pGEM-EC20, they were introduced into DH10B and DH5α strains, similarly to pTE-EC20 and pGEX-EC20 that were introduced into BL21 strain. The EC20 expression was confirmed by SDS-PAGE analysis. The recombinant clones’ resistances to Cd2+ were determined by MIC analyses. The MIC for Cd2+ of DH10B/pBKS-EC20 and DH10B/pGEM-EC20 were 2.5 mM (EC20 induced), and 0.312 mM (EC20 repressed); respectively, 16 and 2 times higher than the control DH10B/pBsKS (0.156 mM). The MIC for Cd2+ of BL21/pTE-EC20 was 10.0 mM (EC20 induced) and 2.5 mM (EC20 repressed), compared with the control BL21/pTE which was only 1.25 mM. Analysis of ICP-AES showed that BL21/pGEX-EC20, after growth on the condition of EC20 expression, absorbed 37.5% of Cd2+, and even when cultured into the non-induction condition of EC20 expression, it absorbed 11.5%. These results allow the conclusion that recombinant E. coli clones expressing the synthetic phytochelatin EC20 show increased capacity for Cd2+ biosorption and enhanced resistance to this toxic ion.

Translate the untranslated: excising as a novel post-splicing event

Maturing of a messenger RNA (mRNA) is a multi-way process producing mRNA variants through diverse splicing events, alternative polyadenylation, RNA editing, etc. Studying posttranscriptional processing of human cold-inducible RNA binding protein (CIRBP), we discovered yet another mechanism that could be added to this list. We named it excising, and it consists of low-accuracy post-splicing deletion of sequence regions of variable length. The main features of the excising process and putative members of corresponding multiprotein machinery were described with a series of cloning vectors and RNA-pulldown assay. Our results highlight a possible role of U-rich stretches and the proteins targeting such motifs in the discovered process. The discovered mechanism suggests the potential translation of 3’-untranslated regions, which may be an adjuvant way of CIRBP activity inhibition or generation of structural and functional diversity.

Establishing a foundation for large DNA transfer to artificial minichromosomes and B insert platforms in maize

In plants, conventional genetic engineering methods limit the number of available traits that could potentially improve the quality of agriculture. Agrobacterium-mediated transformation and biolistic bombardment are tools used in transferring genes into plant cells, both of which result in random integrations into host genomes. The absence of targeting machinery, together with low DNA carrying capacity on most plasmid vectors, limit researchers to a few genes in a single modification experiment, a process that takes [about]1 year in most plant species. While stacking traits from independent genetic modifications allow for an increase in the number of transgenes in a single plant, recovery of all genes in subsequent generations becomes increasingly difficult due to independent segregation in meiosis. Alternatively, the use of binary bacterial artificial chromosomes (BiBACs), large insert cloning vectors, can maintain and transfer up to 300 kps, but are also subject to random integrations. Therefore, establishment of a BiBAC targeting system would be advantageous for researchers focusing on creating plant lines that contain several genes that work together to express complex traits, such as disease resistance clusters or whole biosynthetic pathways. Additionally, BiBAC targeting to a location outside the native chromosomal sets, such as an artificial minichromosome or B chromosome platform, would enable researchers to stack traits without disrupting endogenous sequences.