Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation

Too close for comfort : The role of Contact-Dependent growth Inhibition (CDI) in interbacterial competition and cooperation

Contact-Dependent growth inhibition (CDI) was discovered in 2005 in the E. coli isolate EC93. Since then our knowledge of CDI systems and their impact on bacterial communities have increased exponentially. Yet many biological aspects of CDI systems are still unknown and their impact on complex microbial communities have only just begun to be studied. CDI systems require the function of three proteins; CdiBAI. The outer-membrane transport protein, CdiB, allows for the transportation of the toxin delivery protein CdiA to the cell surface of an inhibitor cell. Through a contact- and receptor-dependent interaction with a target cell the toxic C-terminal domain of CdiA is cleaved off and delivered into the target cell were it mediates a growth arrest. Different CdiA-CT domains encodes for diverse toxic activities, such as nucleases and membrane ionophore toxins. Each unique CdiA-CT toxin has a cognate immunity protein (CdiI) that binds and neutralize against its toxic activity, thus preventing a possible self-inhibition. In this thesis I have studied the effect of CDI system(s) on both single cell and population level, within both intra- and interspecies bacterial communities. The findings presented here shows that multiple class I cdiBAI loci within a cell can function in a synergetic manner and act as versatile interbacterial warfare systems able to inhibit the growth of rival bacteria, even when CdiA expression is low. We also show that class II CdiA receptor-binding domains can mediate broad-range cross-species toxin delivery and growth inhibition, even when a non-optimal target cell receptor is expressed at a low level. Additionally, we show that the cdiA gene supports the expression of two separate proteins. The full-length CdiA protein, able to mediate an extracellular toxin delivery, but also the discrete CdiA-CT toxin domain. This stand-alone CdiA-CT expression was stress-dependent and together with its cognate CdiI immunity protein functioned as a selfish-genetic element. Moreover, we show that CDI systems can increase bacterial stress tolerance via an extracellular toxin delivery between kin-cells. This stress tolerance phenotype only occurred under conditions when we also observed a selective degradation of the CdiI immunity protein. Therefore, we suggest that a selective CdiI degradation allows for a sub-population of cells to self-intoxicate, thereby becoming transiently dormant, which confers an increase in stress tolerance. The findings presented in this thesis collectively suggest that CDI systems could function as a pseudo-quorum sensing system able to mediate behavioral changes and stress tolerance within a sub-population of cells in a bacterial community

Too close for comfort : The role of Contact-Dependent growth Inhibition (CDI) in interbacterial competition and cooperation

Contact-Dependent growth inhibition (CDI) was discovered in 2005 in the E. coli isolate EC93. Since then our knowledge of CDI systems and their impact on bacterial communities have increased exponentially. Yet many biological aspects of CDI systems are still unknown and their impact on complex microbial communities have only just begun to be studied. CDI systems require the function of three proteins; CdiBAI. The outer-membrane transport protein, CdiB, allows for the transportation of the toxin delivery protein CdiA to the cell surface of an inhibitor cell. Through a contact- and receptor-dependent interaction with a target cell the toxic C-terminal domain of CdiA is cleaved off and delivered into the target cell were it mediates a growth arrest. Different CdiA-CT domains encodes for diverse toxic activities, such as nucleases and membrane ionophore toxins. Each unique CdiA-CT toxin has a cognate immunity protein (CdiI) that binds and neutralize against its toxic activity, thus preventing a possible self-inhibition. In this thesis I have studied the effect of CDI system(s) on both single cell and population level, within both intra- and interspecies bacterial communities. The findings presented here shows that multiple class I cdiBAI loci within a cell can function in a synergetic manner and act as versatile interbacterial warfare systems able to inhibit the growth of rival bacteria, even when CdiA expression is low. We also show that class II CdiA receptor-binding domains can mediate broad-range cross-species toxin delivery and growth inhibition, even when a non-optimal target cell receptor is expressed at a low level. Additionally, we show that the cdiA gene supports the expression of two separate proteins. The full-length CdiA protein, able to mediate an extracellular toxin delivery, but also the discrete CdiA-CT toxin domain. This stand-alone CdiA-CT expression was stress-dependent and together with its cognate CdiI immunity protein functioned as a selfish-genetic element. Moreover, we show that CDI systems can increase bacterial stress tolerance via an extracellular toxin delivery between kin-cells. This stress tolerance phenotype only occurred under conditions when we also observed a selective degradation of the CdiI immunity protein. Therefore, we suggest that a selective CdiI degradation allows for a sub-population of cells to self-intoxicate, thereby becoming transiently dormant, which confers an increase in stress tolerance. The findings presented in this thesis collectively suggest that CDI systems could function as a pseudo-quorum sensing system able to mediate behavioral changes and stress tolerance within a sub-population of cells in a bacterial community

Revisionsberättelsens betydelse : En studie om fortlevnadsvarning

Om det finns väsentliga osäkerhetsfaktorer för ett företags fortsatta drift har företagets revisor en skyldighet att uttala sig om det i revisionsberättelsen. ISA 570 är ett internationellt regelverk inom revision som infördes i Sverige år 2004. En översättning gjordes av ISA 570 going concern som i Sverige fick heta fortsatt drift. Det är svårt för revisorer att bedöma och uttala sig om ett företags fortsatta drift då det kan finnas många faktorer som påverkar. Regelverket innehåller ingen bestämd standardutformning för fortlevnadsvarning vilket kan skapa ytterligare osäkerhet hos revisorer. Enligt aktiebolagslagen är revisorer skyldiga att uttala sig om företag är likvidationspliktiga. Ett sådant uttalande kan uppfattas av revisorn som en indirekt varning för företagets fortlevnad. Revisorsnämnden har dock klargjort att det inte anses vara en fortlevnadsvarning. Syftet med den här studien är att undersöka sambandet mellan revisorns uttalande om fortlevnadsvarning och inledd konkurs. Syftet är även att undersöka om det är någon skillnad mellan olika revisionsbyråer/revisorer när det gäller träffsäkerhet i uttalandet om fortlevnad. Undersökningens resultat visar att det finns ett samband mellan inledd konkurs och fortlevnadsvarning. Det visar även att det finns ett samband mellan fortlevnadsvarning och senare inledd konkurs. Ytterligare en slutsats som kan dras utefter resultatet är att de mellanstora revisionsbyråerna har varit mest tillförlitliga när det gäller uttalande om fortlevnad.If there are essential uncertainties for the company's going concern, the auditor are obligated to comment on it in the audit report. ISA 570 is an international regulation of audits that were introduced in Sweden in 2004. A translation was made of ISA 570 going concern for Sweden that was called "fortsatt drift". It is difficult for auditors to assess and comment on the company's going concern as there may be many influencing factors. ISA 570 contain no standard design for warning of going concern, which could create further uncertainty among auditors. According to the Swedish law "Aktiebolagslagen" auditors are obligated to comment if the company is going into liquidation. Such a statement may be seen as an indirect warning about the company's survival. Although the "Revisorsnämnden" in Sweden has made it clear that this is not considered to be a warning of going concern. The purpose of this study is to examine the relationship between the auditor's statement and warning of going concern and initiated bankruptcy. The purpose is also to investigate whether there is any difference between accounting firms / auditors regarding the accuracy of the statement about going concern. The survey results show that there is a relationship between the initiated bankruptcy and warning about going concern. It also showed that there is a correlation between survival warning, and later initiated bankruptcy. Another conclusion is that the mid-sized accounting firms have been the most reliable when it comes to statement about going concern

Revisionsberättelsens betydelse : En studie om fortlevnadsvarning

Om det finns väsentliga osäkerhetsfaktorer för ett företags fortsatta drift har företagets revisor en skyldighet att uttala sig om det i revisionsberättelsen. ISA 570 är ett internationellt regelverk inom revision som infördes i Sverige år 2004. En översättning gjordes av ISA 570 going concern som i Sverige fick heta fortsatt drift. Det är svårt för revisorer att bedöma och uttala sig om ett företags fortsatta drift då det kan finnas många faktorer som påverkar. Regelverket innehåller ingen bestämd standardutformning för fortlevnadsvarning vilket kan skapa ytterligare osäkerhet hos revisorer. Enligt aktiebolagslagen är revisorer skyldiga att uttala sig om företag är likvidationspliktiga. Ett sådant uttalande kan uppfattas av revisorn som en indirekt varning för företagets fortlevnad. Revisorsnämnden har dock klargjort att det inte anses vara en fortlevnadsvarning. Syftet med den här studien är att undersöka sambandet mellan revisorns uttalande om fortlevnadsvarning och inledd konkurs. Syftet är även att undersöka om det är någon skillnad mellan olika revisionsbyråer/revisorer när det gäller träffsäkerhet i uttalandet om fortlevnad. Undersökningens resultat visar att det finns ett samband mellan inledd konkurs och fortlevnadsvarning. Det visar även att det finns ett samband mellan fortlevnadsvarning och senare inledd konkurs. Ytterligare en slutsats som kan dras utefter resultatet är att de mellanstora revisionsbyråerna har varit mest tillförlitliga när det gäller uttalande om fortlevnad.If there are essential uncertainties for the company's going concern, the auditor are obligated to comment on it in the audit report. ISA 570 is an international regulation of audits that were introduced in Sweden in 2004. A translation was made of ISA 570 going concern for Sweden that was called "fortsatt drift". It is difficult for auditors to assess and comment on the company's going concern as there may be many influencing factors. ISA 570 contain no standard design for warning of going concern, which could create further uncertainty among auditors. According to the Swedish law "Aktiebolagslagen" auditors are obligated to comment if the company is going into liquidation. Such a statement may be seen as an indirect warning about the company's survival. Although the "Revisorsnämnden" in Sweden has made it clear that this is not considered to be a warning of going concern. The purpose of this study is to examine the relationship between the auditor's statement and warning of going concern and initiated bankruptcy. The purpose is also to investigate whether there is any difference between accounting firms / auditors regarding the accuracy of the statement about going concern. The survey results show that there is a relationship between the initiated bankruptcy and warning about going concern. It also showed that there is a correlation between survival warning, and later initiated bankruptcy. Another conclusion is that the mid-sized accounting firms have been the most reliable when it comes to statement about going concern

Class II contact‐dependent growth inhibition (CDI) systems allow for broad‐range cross‐species toxin delivery within the Enterobacteriaceae family

Contact‐dependent growth inhibition (CDI) allows bacteria to recognize kin cells in mixed bacterial populations. In Escherichia coli, CDI mediated effector delivery has been shown to be species‐specific, with a preference for the own strain over others. This specificity is achieved through an interaction between a receptor‐binding domain in the CdiA protein and its cognate receptor protein on the target cell. But how conserved this specificity is has not previously been investigated in detail. Here, we show that class II CdiA receptor‐binding domains and their Enterobacter cloacae analog are highly promiscuous, and can allow for efficient effector delivery into several different Enterobacteriaceae species, including Escherichia, Enterobacter, Klebsiella and Salmonella spp. In addition, although we observe a preference for the own receptors over others for two of the receptor‐binding domains, this did not limit cross‐species effector delivery in all experimental conditions. These results suggest that class II CdiA proteins could allow for broad‐range and cross‐species growth inhibition in mixed bacterial populations

Muhasebe ihtiyatlılık kavramı ve İMKB ulusal sınai endeksinde işlem gören işletmeler üzerine bir araştırma

Bu çalışmada, muhasebenin temel kavramlarından olan ihtiyatlılık kavramı üzerinde durulmuş ve istatistiksel yöntemler kullanılarak İMKB ulusal sınai endeksinde işlem gören işletmelerin ihtiyatlı olup olmadıkları ve finansal başarı ile ihtiyatlılık arasında bir ilişkinin olup olmadığı araştırılmıştır. Çalışmanın temelinde ihtiyatlılık kavramının kurumsal yönetim ve muhasebe standartlarının önemli bir ilkesi olduğu ve uzun vadede başarılı işletmelerin aynı zamanda ihtiyatlı da oldukları varsayımı yatmaktadır. Bu doğrultuda İMKB ulusal sınai endeksinde yer alan 28 adet işletmenin 16 yıllık zaman serileri ele alınarak regresyon analizi yapılmıştır ve şu sonuca ulaşılmıştır. 28 işletmeden sadece bir tanesi ihtiyatlıdır ve İMKB ulusal sınai endeksinde işlem gören finansal açıdan başarılı işletmeler ihtiyatlı değildir. Anahtar Kelimeler: İhtiyatlılık, Muhasebe Standartları, Kurumsal Yönetim.\ud In this study, it has been mentioned that conservatism concept which is one of the accounting principles and searched that whether the companies traded in ISE Public Industrial Index are conservative or not, there is a relationship between financial success and conservatism in using statistical methods. It is supposed that in the principal of study conservatism is an important principle of institutional management and accounting standards and successful companies in the long run are also conservative. It has been carried out that regression analysis to 28 companies' traded in ISE Public Industrial Index by using 16 years time series and result is following. Just one of 28 companies is conservative and financially successful companies traded in ISE Public Industrial Index are not conservative. Key Words: Conservatism, Accounting Standards, Institutional Management

RHS-elements function as type II toxin-antitoxin modules that regulate intra-macrophage replication of Salmonella Typhimurium

RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.

Escherichia coli EC93 deploys two plasmid-encoded class I contact-dependent growth inhibition systems for antagonistic bacterial interactions

The phenomenon of contact-dependent growth inhibition (CDI) and the genes required for CDI (cdiBAI) were identified and isolated in 2005 from an Escherichia coli isolate (EC93) from rats. Although the cdiBAIEC93 locus has been the focus of extensive research during the past 15 years, little is known about the EC93 isolate from which it originates. Here we sequenced the EC93 genome and find two complete and functional cdiBAI loci (including the previously identified cdi locus), both carried on a large 127 kb plasmid. These cdiBAI systems are differentially expressed in laboratory media, enabling EC93 to outcompete E. coli cells lacking cognate cdiI immunity genes. The two CDI systems deliver distinct effector peptides that each dissipate the membrane potential of target cells, although the two toxins display different toxic potencies. Despite the differential expression and toxic potencies of these CDI systems, both yielded similar competitive advantages against E. coli cells lacking immunity. This can be explained by the fact that the less expressed cdiBAI system (cdiBAIEC93-2) delivers a more potent toxin than the highly expressed cdiBAIEC93-1 system. Moreover, our results indicate that unlike most sequenced CDI+ bacterial isolates, the two cdi loci of E. coli EC93 are located on a plasmid and are expressed in laboratory media