Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads.

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols.

Overcoming Challenges in Engineering the Genetic Code.

Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code.

Probing the limits of genetic recoding in essential genes.

Engineering radically altered genetic codes will allow for genomically recoded organisms that have expanded chemical capabilities and are isolated from nature. We have previously reassigned the translation function of the UAG stop codon; however, reassigning sense codons poses a greater challenge because such codons are more prevalent, and their usage regulates gene expression in ways that are difficult to predict. To assess the feasibility of radically altering the genetic code, we selected a panel of 42 highly expressed essential genes for modification. Across 80 Escherichia coli strains, we removed all instances of 13 rare codons from these genes and attempted to shuffle all remaining codons. Our results suggest that the genome-wide removal of 13 codons is feasible; however, several genome design constraints were apparent, underscoring the importance of a strategy that rapidly prototypes and tests many designs in small pieces.

Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering.

Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.

Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate.

The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5' ends of double-stranded DNA are recessed by λ exonuclease, leaving behind 3' overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinase-catalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.

The effect of a shortened dry period on intramammary infections during the subsequent lactation.

Several recent studies have investigated the effect of shortened dry periods on milk production in the subsequent lactation. What is lacking from these studies is an understanding of the effect that a shortened dry period has on udder health. Four herds, 156 cows, were studied to determine if a shortened dry period (30 d) had a negative effect on mammary gland health during the subsequent lactation as opposed to cows assigned to a long, 45 or 60 d, dry period. Cows in 2 herds were assigned to either 30- or 60-d dry periods (group I), whereas cows in the other 2 herds were assigned to either 30- or 45-d dry periods (group II). Intramammary instillation of commercial preparations of cephapirin benzathine, 300 mg (dry cow formulation), was given to cows assigned a 45- or 60-d dry period length protocol, and 200 mg (lactating cow formulation) was administered to cows assigned a 30-d dry period. Differences in response variables to dry period length were compared within group. Cure rates for 60- vs. 30-d dry period cows were 72% (28/39) vs. 81% (30/37) and 74% (25/34) and 73% (27/37) for 45- vs. 30-d dry periods. Differences were not statistically significant for either comparison group. The majority of intramammary infections were caused by the minor pathogens, coagulase-negative staphylococci (n = 102) or Corynebacterium bovis (n = 11). Only 11 cows had intramammary infections by major pathogens. The herd average percentage of new intramammary infections ranged from 6 to 9% and did not differ among herds between treatment groups. Linear somatic cell counts were not significantly affected by dry period length during the first 6 to 7 mo of the subsequent lactation. Milk production did differ between groups. Mature equivalent milk production was greater in group I cows given a 60-d dry period (11,942 +/- 2,059 kg) as opposed to those given a 30-d dry period (10,749 +/- 2,321 kg). Cows given a 45-d dry period did not produce more milk than cows with a 30-d dry period in group II. Although shortening the dry period to 30 d did not have untoward effects on mammary gland health as measured by intramammary infections or milk somatic cell counts, production may be adversely affected when dry periods are shortened to 30 d.

Quantitative monitoring by polymerase colony assay of known mutations resistant to ABL kinase inhibitors.

Resistance to molecularly targeted chemotherapy, and the development of novel agents that are active against resistant forms of target proteins create the need for a sensitive and quantitative assay to monitor drug-resistant mutations in patients to guide treatment and assess response. Here, we describe an application of the polymerase colony (polony) method to identify and quantify known point mutations in the BCR-ABL oncogene in patients with chronic myelogenous leukemia who evolve resistance to ABL kinase inhibitors. The assay can detect mutations with a sensitivity of 10(-4), quantify the burden of drug-resistant cells, and simultaneously monitor the dynamics of several coexisting mutations. As a proof of concept, we analysed blood samples from three patients undergoing therapy with ABL kinase inhibitors and found that the patients' response to therapy correlated with our molecular monitoring. We were also able to detect mutations emerging in patients long before clinical relapse. Therefore, the polony assay could be applied to a larger patient sample to assess the utility of early mutation detection in patient-specific treatment decisions. Finally, this methodology could be a valuable research tool to shed light on the natural behavior of mutations pre-existing kinase inhibitors therapy and either disappearing over time or slowly taking over.

Transfusion-related acute lung injury in the paediatric patient: Two case reports and a review of the literature.

Transfusion-related acute lung injury (TRALI) is increasingly recognized as a major complication of transfusion therapy; it was the leading cause of transfusion-related fatalities in the United States in 2003. Most cases of TRALI that have been reported are in adult patients. We present two cases of TRALI that occurred in children and review the existing literature of paediatric TRALI. The paediatric TRALI case reports highlight two laboratory findings that can help in the diagnosis of TRALI: transient leucopenia and an elevated pulmonary oedema fluid/plasma protein ratio. These two simple diagnostic tests can help rule out other diagnoses and add confidence to the clinical diagnosis of TRALI. Finally, our first case also highlights the potential danger of directed maternal blood donations, which may increase the risk of paediatric TRALI.

First Report of the Root-Knot Nematode Meloidogyne floridensis on Tomato (Lycopersicon esculentum) in Florida.

The state of Florida is the largest producer of fresh market tomato (Lycopersicon esculentum L.) in the United States with 2003 yields of 634 million kg on 17,700 ha valued at 516 million dollars. Effective crop management is essential for production of vegetables in Florida because of the presence of intense pest pressure. The identification of the pests present is the first step in the development of a successful IPM (integrated pest management) program. Root-knot nematodes (Meloidogyne spp.) are common nematodes that parasitize vegetables in Florida and cause significant yield reductions when not properly managed. In 2003 field experiments, soil was collected from two research farms in Saint Lucie and Seminole counties in Florida. Galling caused by root-knot nematode was observed on tomato at both locations. Since females suitable for identification are difficult to obtain from field-grown roots, field soil was placed in pots in the greenhouse and planted with Lycopersicon esculentum cv. Rutgers. Standard morphological techniques, differential host tests, and isozyme phenotypes were used in nematode identification. Female root-knot nematodes were extracted from tomato roots and placed in extraction buffer (10% wt/vol sucrose, 2% vol/vol Triton X-100, 0.01% wt/vol bromophenol blue). The females were crushed, loaded on a polyacrylamide gel, and separated by electrophoresis using the PhastSystem (Amersham Biosciences, Piscataway, NJ). The activities of malate dehydrogenase and esterase enzymes were detected using standard techniques. Isozyme phenotypes consistent with Meloidogyne incognita (Kofoid and White) Chitwood and M. javanica (Treub) Chitwood as well as with the newly described M. floridensis Handoo (1) were observed at both locations. To our knowledge, this is the first report of M. floridensis naturally occurring on tomato in Florida. The identification and distribution of M. floridensis in vegetable production fields is important for disease management throughout the state since the host range is likely different from other Meloidogyne spp. Reference: (1) Z. A. Handoo et al. J. Nematol. 36:20, 2004.