Reduced mutation rate and increased transformability of transposon-free Acinetobacter baylyi ADP1-ISx

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting “clean-genome” ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. </jats:p

Heterodimeric Transcription Factors in a Yeast One-Hybrid Assay

The yeast one-hybrid (Y1H) system is an important and powerful assay for the detection of protein-DNA interactions. However, the system cannot assay for heterodimeric transcription factors (TFs), key regulators of spatiotemporal gene expression. A modified Y1H system was developed using novel plasmid constructs to coexpress Gal4 activation domain (AD)-fusion heterodimer pairs in existing and newly constructed bait reporter yeast strains. The clones and strategies developed provide a foundation to effectively screen dimerizing TFs for sequence specific interactions

An Assessment of Fire Safety in Australia’s International Student Housing

An assessment of international student awareness in fire safety issues with respect to their housing in Australia was performed by this project, which was commissioned by the Australasian Fire and Emergency Services Authorities Council. As a basis to characterize this situation, pieces of fire safety legislation, education materials, and techniques for multicultural pedagogy were gathered and analyzed. A survey was administered to collect data from a representative population of students throughout Australia. Lapses in fire safety knowledge and compliance made apparent through the research were addressed in FireAway, an education plan, and legislative recommendations

Predicting the Genetic Stability of Engineered DNA Sequences with the EFM Calculator

Unwanted evolution can rapidly degrade the performance of genetically engineered circuits and metabolic pathways installed in living organisms. We created the Evolutionary Failure Mode (EFM) Calculator to computationally detect common sources of genetic instability in an input DNA sequence. It predicts two types of mutational hotspots: deletions mediated by homologous recombination and indels caused by replication slippage on simple sequence repeats. We tested the performance of our algorithm on genetic circuits that were previously redesigned for greater evolutionary reliability and analyzed the stability of sequences in the iGEM Registry of Standard Biological Parts. More than half of the parts in the Registry are predicted to experience >100-fold elevated mutation rates due to the inclusion of unstable sequence configurations. We anticipate that the EFM Calculator will be a useful negative design tool for avoiding volatile DNA encodings, thereby increasing the evolutionary lifetimes of synthetic biology devices

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy

Observation of WWWWWW Production in pppp Collisions at s\sqrt s =13 TeV with the ATLAS Detector

International audienceThis Letter reports the observation of $WWW$ production and a measurement of its cross section using 139 fb$^{-1}$ of proton-proton collision data recorded at a center-of-mass energy of 13 TeV by the ATLAS detector at the Large Hadron Collider. Events with two same-sign leptons (electrons or muons) and at least two jets, as well as events with three charged leptons, are selected. A multivariate technique is then used to discriminate between signal and background events. Events from $WWW$ production are observed with a significance of 8.0 standard deviations, where the expectation is 5.4 standard deviations. The inclusive $WWW$ production cross section is measured to be $820 \pm 100\,\text{(stat)} \pm 80\,\text{(syst)}$ fb, approximately 2.6 standard deviations from the predicted cross section of $511 \pm 18$ fb calculated at next-to-leading-order QCD and leading-order electroweak accuracy