Transformation ofBurkholderia pseudomalleiby Electroporation

Mack, Kerri; Titball, Richard W.

doi:10.1006/abio.1996.0430

Cited by 21 publications

(12 citation statements)

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“…Unfortunately, all three proved too large for Qβ, as evidenced by poor infectivity and/or plaque formation, even when fused to full-length A1 or A1 3′ truncation. The transformation efficiency of these constructs dropped with the increasing size of the insert but could be somewhat improved by using the rubidium chloride method with heat shock at 43.5°C, in contrast to conventional methods [32]. The Qβ phage remains functional (able to absorb and infect) with up to 60 nucleotides inserted into the A1 gene.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

2014

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The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

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Section: Discussionmentioning

confidence: 99%

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

2014

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“…Genomic DNA was isolated from B. pseudomallei as previously described [22]. Restriction endonucleases and DNA modifying enzymes (New England Biolabs) were used as per manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Predicted Trimeric Autotransporter Adhesin Required for Biofilm Formation of Burkholderia pseudomallei

et al. 2013

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The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A.

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“…Based on comparison with five studies, correlation predictions of peak transfection conditions in vivo [39][40][41][42][43] were of similar accuracy as for cell suspensions (MAPE = 51 ± 53%), which suggests that this correlation may be of importance in developing clinical applications. Predictions of peak transfection conditions for bacteria and yeast 22, [44][45][46][47][48] (MAPE = 48 ± 62%) were not as good as for mammalian cells, but predictions of 50% viability conditions were similarly accurate (MAPE = 21 ± 8%) (eg Figure 2d).…”

Section: Mammalian Viability Predictionsmentioning

confidence: 71%