A new capillary electrophoresis interface to electrospray ionization mass spectrometry (CE/ESI-MS) is introduced in which the electrical connection to the CE capillary outlet/ESI electrode is achieved by transfer of small ions related to the background electrolyte (BGE) through a porous section near the CE capillary outlet. In this design, only a small section of the capillary wall is made porous. The porous section is created by first thinning a small section of the capillary wall by drilling a well into it and then etching the remaining thin wall porous. This design has two advantages over previous designs (in which the whole circumference of the capillary was made porous): first, the capillary interface is more robust because only a small section of it is made porous, and therefore, no liquid junction is needed to secure the porous section. The electrical connection is achieved simply by inserting the capillary outlet containing the porous junction into the existing ESI needle and filling the needle with the BGE. Second, the time required to make the fused silica porous is reduced from approximately 1 h to a few minutes. In addition, there is no dead volume associated with the porous design, and because the actual metal/liquid contact occurs outside of the CE capillary, bubble formation due to redox reactions of water at the electrode does not affect CE/ESI-MS performance. The performance of this interface is demonstrated by the analyses of peptide and protein mixtures.

A general bacterial genome engineering framework, ‘Genome Editing via Targetrons and Recombinases' (GETR), is presented. GETR combines mobile group II introns (targetrons) and the Cre/lox system to allow genomic manipulations at a large scale.

The mobile Lactococcus lactis Ll.LtrB group II intron integrates into DNA target sites by a mechanism in which the intron RNA reverse splices into one DNA strand, while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end to prime reverse transcription of the inserted intron RNA. These reactions are mediated by an RNP particle that contains the intron-encoded protein and the excised intron lariat RNA, with both the protein and base pairing of the intron RNA used to recognize DNA target sequences. Here, computational analysis indicates that Escherichia coli DNA target sequences that support Ll.LtrB integration have greater predicted bendability than do random Escherichia coli genomic sequences, and atomic force microscopy shows that target DNA is bent during the reaction with Ll.LtrB RNPs. Time-course and mutational analyses show that DNA bending occurs after reverse splicing and requires subsequent interactions between the intron-encoded protein and the 3'-exon, which lead to two progressively larger bend angles. Our results suggest a model in which RNPs bend the target DNA by maintaining initial contacts with the 5' exon, while engaging in subsequent 3'-exon interactions that successively position the scissile phosphate for bottom-strand cleavage at the DNA endonuclease active site and then reposition the 3' end of the cleaved bottom strand at the reverse transcriptase active site for initiation of cDNA synthesis. Our findings indicate that bendability of the DNA target site is a significant factor for Ll.LtrB RNP integration.Mobile group II introns, which are found in bacterial and organelle genomes, are retroelements that integrate site-specifically ("retrohome") into DNA target sites at high frequency and retrotranspose to ectopic sites that resemble the normal homing site at low frequency (reviewed in refs (1-4)). The integration reactions are mediated by an RNP complex that is formed during RNA splicing and contains the intron-encoded protein (IEP) 1 and the excised intron lariat RNA (3,4). RNPs initiate intron mobility by recognizing relatively long (30−35 bp) DNA target sites, with both the IEP and base pairing of the intron RNA contributing to the recognition of DNA † This work was supported by NIH grant GM37949 and Welch Foundation Grant F-1607 to A.M.L. and by support from the Welch NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript target sequences (5-8). Then, the intron RNA reverse splices into one strand of the DNA and is reverse transcribed by the IEP, yielding an intron cDNA that is integrated into the recipient genome by host DNA recombination or repair mechanisms (reviewed in refs 3,4). Their very high integration frequencies and target specificity combined with the ability to program insertion into different target sites by modifying the base-pairing sequences of the intron RNA have made it possible to develop mobile group II introns into highly efficient bacterial gene targeting vec...

BackgroundAnthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome.ResultsGroup II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion.ConclusionsThe design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics.