Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955

Background The thermophile Geobacillus thermoglucosidasius has considerable attraction as a chassis for the production of chemicals and fuels. It utilises a wide range of sugars and oligosaccharides typical of those derived from lignocellulose and grows at elevated temperatures. The latter improves the rate of feed conversion, reduces fermentation cooling costs and minimises the risks of contamination. Full exploitation of its potential has been hindered by a dearth of effective gene tools. Results Here we designed and tested a collection of vectors (pMTL60000 series) in G. thermoglucosidasius NCIMB 11955 equivalent to the widely used clostridial pMTL80000 modular plasmid series. By combining a temperature-sensitive replicon and a heterologous pyrE gene from Geobacillus kaustophilus as a counter-selection marker, a highly effective and rapid gene knock-out/knock-in system was established. Its use required the initial creation of uracil auxotroph through deletion of pyrE using allele-coupled exchange (ACE) and selection for resistance to 5-fluoroorotic acid. The turnaround time for the construction of further mutants in this pyrE minus strain was typically 5 days. Following the creation of the desired mutant, the pyrE allele was restored to wild type, within 3 days, using ACE and selection for uracil prototrophy. Concomitant with this process, cargo DNA (pheB) could be readily integrated at the pyrE locus. The system’s utility was demonstrated through the generation in just 30 days of three independently engineered strains equivalent to a previously constructed ethanol production strain, TM242. This involved the creation of two in-frame deletions (ldh and pfl) and the replacement of a promoter region of a third gene (pdh) with an up-regulated variant. In no case did the production of ethanol match that of TM242. Genome sequencing of the parental strain, TM242, and constructed mutant derivatives suggested that NCIMB 11955 is prone to the emergence of random mutations which can dramatically affect phenotype. Conclusions The procedures and principles developed for clostridia, based on the use of pyrE alleles and ACE, may be readily deployed in G. thermoglucosidasius. Marker-less, in-frame deletion mutants can be rapidly generated in 5 days. However, ancillary mutations frequently arise, which can influence phenotype. This observation emphasises the need for improved screening and selection procedures at each step of the engineering processes, based on the generation of multiple, independent strains and whole-genome sequencing

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

Thermal Conductivity of Cryoprotective Agents with Applications to Cryopreservation by Vitrification

<p>Cryopreservation is the preservation of biomaterials at extremely low temperatures. It is the only alternative for long-term storage of high quality biomaterials, with applications to biobanking and transplant medicine. Cryopreservation success revolves around the control of ice formation, which is known to be harmful. Ice formation is a path-dependent phenomenon, affected by the thermal history and presence of nucleation promotors. Cryoprotective agents (CPAs) are commonly added to the biomaterial to be preserved, in order to suppress ice formation and inhibit its growth during the cryopreservation protocol. Ice-free cryopreservation can be achieved in large-size systems when the biomaterial is loaded with a high CPA concentration solution and cooled rapidly, in a process that is known as vitrification (vitreous means glassy in Latin). During vitrification, the CPA viscosity increases exponentially with decreasing temperature, while the material is cooled to deep cryogenic temperatures faster than the typical time scale for crystallization. The material can potentially be stored indefinitely at such low temperatures. Large-size vitrification is associated with three competing needs on the CPA concentration. Since the cooling rate at the center of the specimen decreases with the increasing specimen size due to the scaling conductive resistance, higher CPA concentrations may be required to suppress crystallization in larger specimens. Higher CPA concentration generally requires lower cooling rates to avoid ice crystallization. On the other hand, since CPAs are potentially toxic, the lowest possible CPA concentration is required to maintain viability and facilitate functional recovery. The decrease in CPA concentration combined with an increase in cooling rates may intensify thermo-mechanical stress due to non-uniform thermal contraction to the point of structural destruction. Essentially, successful cryopreservation represents the outcome of an optimization problem on the composition and concentration of the CPA cocktail. The work presented in this thesis combines an experimental study on the thermal conductivity of relevant materials, and a theoretical study to identify the effects of the measured values on cryopreservation protocols. The unique contributions presented as the initial stage of the experimental study are: (i) the modification of the cryomacroscope and creation of an experimental program to make thermal conductivity measurements of CPA based on the existing transient hot wire technique, (ii) to develop a protocol for making thermal conductivity measurements during rewarming portion of the cryoprotocol, and (iii), to begin generating a data bank of thermal conductivity of CPA and materials used in cryopreservation. Thermal conductivity measurements are presented for the CPA Dimethyl Sulfoxide (DMSO), over a concentration range of 2M to 10M, in a temperature range of -180°C to 25°C. Samples of 2M to 6M DMSO were found to crystallize at quasi-steady cooling rates, while samples of 7.05 to 10M were found to vitrify. Thermal conductivities of the crystallized and vitrified material reach a tenfold difference at -180°C. The quality of measurements using the presented technique has been verified theoretically by means of finite element analysis (FEA) using the commercial code ANSYS. This experimental study is expanded to the study of thermal conductivity of the CPA cocktail DP6--a mixture of 3M DMSO and 3M propylene glycol, which has drawn significant attention in the cryobiology community in recent times. The unique contributions are the first thermal conductivity measurements reported in literature of the combined effect of DP6 with synthetic ice modulators (SIMs), including 6% 1,3Cyclohexanediol, 6% 2,3Butanediol, and 12% PEG400. Results of this study demonstrate that the thermal conductivity may vary by three fold between the amorphous and crystalline phases of DP6 below the glass transition temperature. Results of this study further demonstrate the ability of SIMs to decrease the extent of crystallization in DP6, even at subcritical cooling and rewarming rates. The accompanying theoretical investigation focuses on cryopreservation in a kidney model, in effort to explore how the thermal history is affected by variations in the measured thermal conductivity. This analysis is based on FEA using the commercial code ANSYS. In particular, the unique contributions of this study are: (i) thermal analysis of a vitrifying rabbit kidney based on an established rabbit-kidney cryopreservation protocol, and (ii), exploring scale-up thermal effects to a human-size organ. This represents a 21-fold increase in organ size. Results indicate that even in the case of the human kidney, cooling rates remain high enough in all parts of the kidney to prevent ice formation at temperatures above -100oC.</p

Large Thermal Conductivity Differences between the Crystalline and Vitrified States of DMSO with Applications to Cryopreservation.

Thermal conductivity of dimethyl-sulfoxide (DMSO) solution is measured in this study using a transient hot wire technique, where DMSO is a key ingredient in many cryoprotective agent (CPA) cocktails. Characterization of thermal properties of cryoprotective agents is essential to the analysis of cryopreservation processes, either when evaluating experimental data or for the design of new protocols. Also presented are reference measurements of thermal conductivity for pure water ice and glycerol. The thermal conductivity measurement setup is integrated into the experimentation stage of a scanning cryomacroscope apparatus, which facilitates the correlation of measured data with visualization of physical events. Thermal conductivity measurements were conducted for a DMSO concentration range of 2M and 10M, in a temperature range of -180°C and 25°C. Vitrified samples showed decreased thermal conductivity with decreasing temperature, while crystalline samples showed increased thermal conductivity with decreasing temperature. These different behaviors result in up to a tenfold difference in thermal conductivity at -180°C. Such dramatic differences can drastically impact heat transfer during cryopreservation and their quantification is therefore critical to cryobiology

Large Thermal Conductivity Differences between the Crystalline and Vitrified States of DMSO with Applications to Cryopreservation.

<p>Thermal conductivity of dimethyl-sulfoxide (DMSO) solution is measured in this study using a transient hot wire technique, where DMSO is a key ingredient in many cryoprotective agent (CPA) cocktails. Characterization of thermal properties of cryoprotective agents is essential to the analysis of cryopreservation processes, either when evaluating experimental data or for the design of new protocols. Also presented are reference measurements of thermal conductivity for pure water ice and glycerol. The thermal conductivity measurement setup is integrated into the experimentation stage of a scanning cryomacroscope apparatus, which facilitates the correlation of measured data with visualization of physical events. Thermal conductivity measurements were conducted for a DMSO concentration range of 2M and 10M, in a temperature range of -180°C and 25°C. Vitrified samples showed decreased thermal conductivity with decreasing temperature, while crystalline samples showed increased thermal conductivity with decreasing temperature. These different behaviors result in up to a tenfold difference in thermal conductivity at -180°C. Such dramatic differences can drastically impact heat transfer during cryopreservation and their quantification is therefore critical to cryobiology.</p

Solid fraction during solidification of a water-DMSO mixture, extracted from a phase diagram [13].

<p>Solid fraction during solidification of a water-DMSO mixture, extracted from a phase diagram [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125862#pone.0125862.ref013" target="_blank">13</a>].</p

Thermal conductivity as a function of solid fraction for DMSO at various concentrations, where the Bruggeman model is calculated with Eq (7) for 6M DMSO.

<p>Thermal conductivity as a function of solid fraction for DMSO at various concentrations, where the Bruggeman model is calculated with Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125862#pone.0125862.e007" target="_blank">7</a>) for 6M DMSO.</p

Schematic illustration of the scanning cryomacroscope setup and peripheral instrumentation [11]; the modified experimentation stage for thermal conductivity measurements is displayed with more detail in Fig 2.

<p>Schematic illustration of the scanning cryomacroscope setup and peripheral instrumentation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125862#pone.0125862.ref011" target="_blank">11</a>]; the modified experimentation stage for thermal conductivity measurements is displayed with more detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125862#pone.0125862.g002" target="_blank">Fig 2</a>.</p

Cryomacroscope images of samples in various states: (a) a vitrified 7.05M DMSO sample at a temperature of -147°C; (b) a 2M DMSO sample undergoing crystallization in the form of dendrites at temperature of -10°C; (c) a partially crystallized 6M DMSO sample at a temperature of -58°C; and (d) a completely crystallized 6M DMSO solution at a temperature of -65°C.

<p>Cryomacroscope images of samples in various states: (a) a vitrified 7.05M DMSO sample at a temperature of -147°C; (b) a 2M DMSO sample undergoing crystallization in the form of dendrites at temperature of -10°C; (c) a partially crystallized 6M DMSO sample at a temperature of -58°C; and (d) a completely crystallized 6M DMSO solution at a temperature of -65°C.</p