Exploring DNA-protein interactions on the single DNA molecule level using nanofluidic tools

DNA-protein interactions are at the core of the cellular machinery and single molecule methods have revolutionized the possibilities to study, and our understanding of these interactions on the molecular level. Nanofluidic channels have been extensively used for studying single DNA molecules during the last twelve years and in this review, we discuss how this experimental platform has been extended to studies of DNA-protein interactions. We first present how the design of the device can be tailored for the specific DNA-protein system studied and how the channels can be passivated to avoid non-specific binding of proteins. We then focus on describing the different kinds of DNA-interacting proteins that have been studied in nanofluidic devices, including proteins that compact DNA and proteins that form filaments on DNA. Our main objective is to highlight the diverse functionalitiesof DNA-protein systems that have been characterized using nanofluidic structures and hence demonstrate the versatility of these experimental tools. We finally discuss potential future directions studies of DNA-protein complexes in nanochannels might take, including specific DNA-protein systems that are difficult to analyze with traditional techniques, devices with increased complexity, and fully integrated lab-on-a-chip devices for analysis of material extracted from (single) cells

The word problem for one-relation monoids: a survey

This survey is intended to provide an overview of one of the oldest and most celebrated open problems in combinatorial algebra: the word problem for one-relation monoids. We provide a history of the problem starting in 1914, and give a detailed overview of the proofs of central results, especially those due to Adian and his student Oganesian. After showing how to reduce the problem to the left cancellative case, the second half of the survey focuses on various methods for solving partial cases in this family. We finish with some modern and very recent results pertaining to this problem, including a link to the Collatz conjecture. Along the way, we emphasise and address a number of incorrect and inaccurate statements that have appeared in the literature over the years. We also fill a gap in the proof of a theorem linking special inverse monoids to one-relation monoids, and slightly strengthen the statement of this theorem

The word problem and combinatorial methods for groups and semigroups

The subject matter of this thesis is combinatorial semigroup theory. It includes material, in no particular order, from combinatorial and geometric group theory, formal language theory, theoretical computer science, the history of mathematics, formal logic, model theory, graph theory, and decidability theory. In Chapter 1, we will give an overview of the mathematical background required to state the results of the remaining chapters. The only originality therein lies in the exposition of special monoids presented in §1.3, which uni.es the approaches by several authors. In Chapter 2, we introduce some general algebraic and language-theoretic constructions which will be useful in subsequent chapters. As a corollary of these general methods, we recover and generalise a recent result by Brough, Cain & Pfei.er that the class of monoids with context-free word problem is closed under taking free products. In Chapter 3, we study language-theoretic and algebraic properties of special monoids, and completely classify this theory in terms of the group of units. As a result, we generalise the Muller-Schupp theorem to special monoids, and answer a question posed by Zhang in 1992. In Chapter 4, we give a similar treatment to weakly compressible monoids, and characterise their language-theoretic properties. As a corollary, we deduce many new results for one-relation monoids, including solving the rational subset membership problem for many such monoids. We also prove, among many other results, that it is decidable whether a one-relation monoid containing a non-trivial idempotent has context-free word problem. In Chapter 5, we study context-free graphs, and connect the algebraic theory of special monoids with the geometric behaviour of their Cayley graphs. This generalises the geometric aspects of the Muller-Schupp theorem for groups to special monoids. We study the growth rate of special monoids, and prove that a special monoid of intermediate growth is a group

Utveckling av metod för att bedöma behovet av ogräsbekämpning i spår

En miljöanpassad och resurseffektiv hantering av ogräs i spår ställer krav på god kunskap om vegetationsförhållanden för att behovsanpassa bekämpningsinsatserna. En automatiserad registrering skulle kunna utgöra ett komplement till dagens manuella inspektioner och skulle över tiden helt eller delvis kunna ersätta dessa. En utmaning är att hitta en metod som ger en rimlig upplösning i informationen som samlas in, så att den kan hanteras rationellt av berörda aktörer och samtidigt utgöra ett beslutsunderlag med tillräcklig precision. Projektet studerade två automatiserade metoder som kan vara aktuella för Trafikverket att använda i framtiden: 1) Machine vision metoden utnyttjar kamerasensorer för att känna av sin omgivning i det synliga respektive nära infraröda spektrumet. 2) N-sensorn sänder ut ljus inom det område som reflekteras av växternas klorofyll. Mängden klorofyll ger ett mätvärde som kan korreleras till biomassan. Valet av teknik beror på vad informationen ska användas till. Om syftet är att översiktligt kartlägga vegetationsförekomst i spår, för att planera åtgärder för underhåll, kan N-sensortekniken vara lämplig. Om man över ytan och tiden vill övervaka och kartlägga aktuell och precis vegetationsstatus, för att kunna bekämpa utvald vegetation med rätt insats, är machine vision tekniken bättre lämpad. Såväl machine vision metoden som N-sensortekniken bygger på registrering av data tillsammans med en GPS-positionering. På sikt kan denna information läggas i databaser som är direkt åtkomliga för berörda organisationer och t o m online i fält under eller i samband med en bekämpningsåtgärd. De två teknikerna jämfördes med manuella (visuella) skattningar av ogräsförekomsten. Den visuella skattningen av yttäckningsgrad av ogräs i fält skiljde sig statistiskt mellan olika bedömare. När det gäller att uppskatta frekvensen (antalet) vedartade växter (träd och buskar) inom provytorna så var observatörerna relativt överens. Samma person är ofta konsekvent i sitt bedömande, men att jämföra med andra personers bedömning kan ge missvisande resultat. Systemet för användning av informationen om ogräsförekomst behöver utvecklas som helhet. Tröskelvärden för hur mycket ogräs som kan tolereras på olika typer av spår/driftsplatser är en viktig komponent i ett sådant system. Klassificeringssystemet ska kunna hantera de krav som ställs för att säkerställa banans kvalitet och olika förutsättningar som trafikförhållanden, platsgivna förutsättningar för banan och vegetationens egenskaper. Projektet rekommenderar Trafikverket att: diskutera hur tröskelvärden för vegetationsförekomst på spår kan fastställas genomföra registrering av vegetationsförekomst över längre och fler sträckor med en eller flera av de metoder som studerats i projektet inleda införande av system som effektivt kopplar informationen om vegetation till position inkludera förekomst av vegetation i den registrering som idag sker av spårens (banans) tekniska kvalitet och ansluta datamaterialet till övriga underhållsrelaterade databaser inrätta ett antal representativa ytor där ogräsfloran på spåren regelbundet inventeras och mäts för att få en bild av den långsiktiga utveckling som grund för säkrare prognoser för vegetationsutveckling säkerställa att nödvändiga utbildningsinsatser genomför

Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples

Identification and DNA annotation of a plasmid isolated from Chromobacterium violaceum

Chromobacterium violaceum is a f-proteobacterium found widely worldwide with important biotechnological properties and is associated to lethal sepsis in immune-depressed individuals. In this work, we report the discover, complete sequence and annotation of a plasmid detected in C. violaceum that has been unnoticed until now. We used DNA single-molecule analysis to confirm that the episome found was a circular molecule and then proceeded with NGS sequencing. After DNA annotation, we found that this extra-chromosomal DNA is probably a defective bacteriophage of approximately 44 kilobases, with 39 ORFs comprising, mostly hypothetical proteins. We also found DNA sequences that ensure proper plasmid replication and partitioning as well as a toxin addiction system. This report sheds light on the biology of this important species, helping us to understand the mechanisms by which C. violaceum endures to several harsh conditions. This discovery could also be a first step in the development of a DNA manipulation tool in this bacterium