Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

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Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

10.1101/754242 ◽

2019 ◽

Author(s):

Gabriel A. Suárez ◽

Kyle R. Dugan ◽

Brian A. Renda ◽

Sean P. Leonard ◽

Lakshmi S. Gangavarapu ◽

...

Keyword(s):

Gene Knockout ◽

Single Gene ◽

Golden Gate ◽

Acinetobacter Baylyi ◽

Gene Deletions ◽

Multiple Gene ◽

A Genome ◽

Strain Collection ◽

Acinetobacter Baylyi Adp1 ◽

Improved Methods

ABSTRACTOne goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

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Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis

Journal of Experimental Botany ◽

10.1093/jxb/eraa316 ◽

2020 ◽

Vol 71 (19) ◽

pp. 5963-5975 ◽

Cited By ~ 2

Author(s):

Panupon Khumsupan ◽

Marta A Kozlowska ◽

Douglas J Orr ◽

Andreas I Andreou ◽

Naomi Nakayama ◽

...

Keyword(s):

Gene Knockout ◽

Catalytic Efficiency ◽

Small Subunit ◽

Multiple Gene ◽

Functional Roles ◽

Knockout Mutants ◽

Dna Insertion ◽

Low Levels ◽

Rbcs Genes ◽

Viable Approach

Abstract The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

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Microbial Gutta-Percha Degradation Shares Common Steps with Rubber Degradation by Nocardia nova SH22a

Applied and Environmental Microbiology ◽

10.1128/aem.03016-12 ◽

2012 ◽

Vol 79 (4) ◽

pp. 1140-1149 ◽

Cited By ~ 11

Author(s):

Quan Luo ◽

Sebastian Hiessl ◽

Anja Poehlein ◽

Alexander Steinbüchel

Keyword(s):

Plasmid Dna ◽

Cell Envelope ◽

Degradation Mechanism ◽

Degradation Pathways ◽

Genetic Studies ◽

Content Type ◽

Gutta Percha ◽

Optimized Protocol ◽

Rubber Degradation

ABSTRACTNocardia novaSH22a, a bacterium capable of degrading gutta-percha (GP) and natural rubber (NR), was used to investigate the GP degradation mechanism and the relations between the GP and NR degradation pathways. For this strain, a protocol of electroporation was systematically optimized, and an efficiency of up to 4.3 × 107CFU per μg of plasmid DNA was achieved. By applying this optimized protocol toN. novaSH22a, a Tn5096-based transposon mutagenesis library of this bacterium was constructed. Among about 12,000 apramycin-resistant transformants, we identified 76 stable mutants defective in GP or NR utilization. Whereas 10 mutants were specifically defective in GP utilization, the growth of the other 66 mutants was affected on both GP and NR. This indicated that the two degradation pathways are quite similar and share many common steps. The larger number of GP-degrading defective mutants could be explained in one of two ways: either (i) the GP pathway is more complex and harbors more specific steps or (ii) the steps for both pathways are almost identical, but in the case of GP degradation there are fewer enzymes involved in each step. The analysis of transposition loci and genetic studies on interesting genes confirmed the crucial role of an α-methylacyl-coenzyme A racemase in the degradation of both GP and NR. We also demonstrated the probable involvement of enzymes participating in oxidoreduction reactions, β-oxidation, and the synthesis of complex cell envelope lipids in the degradation of GP.

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One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs

Cell Research ◽

10.1038/cr.2017.81 ◽

2017 ◽

Vol 27 (7) ◽

pp. 933-945 ◽

Cited By ~ 77

Author(s):

Erwei Zuo ◽

Yi-Jun Cai ◽

Kui Li ◽

Yu Wei ◽

Bang-An Wang ◽

...

Keyword(s):

Knockout Mice ◽

Gene Editing ◽

Gene Knockout ◽

Gene Knockout Mice ◽

One Step ◽

Step Generation

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Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus

Analytical and Bioanalytical Chemistry ◽

10.1007/s00216-019-02254-7 ◽

2019 ◽

Vol 412 (2) ◽

pp. 449-462 ◽

Cited By ~ 2

Author(s):

Dania Awad ◽

Thomas Brueck

Keyword(s):

Protein Extraction ◽

Feedback Regulation ◽

Genetic Optimization ◽

Physiochemical Properties ◽

High Lipid ◽

Extraction Buffer ◽

Optimized Protocol ◽

Significant Step ◽

One Step ◽

Purification Methods

AbstractIn the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids.

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Efficient circular gene knockout system for Burkholderiales strain DSM 7029 and Mycobacterium smegmatis mc2 155

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmz054 ◽

2019 ◽

Vol 51 (7) ◽

pp. 697-706 ◽

Cited By ~ 1

Author(s):

Xiaolai Lei ◽

Qiuxia Fan ◽

Tian Huang ◽

Haiyun Liu ◽

Guoping Zhao ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Gene Knockout ◽

Gene Cassette ◽

Integrase Gene ◽

Multiple Gene ◽

System A ◽

Attachment Sites ◽

Sacb Gene ◽

Gene Knockouts ◽

Serine Integrase

Abstract Multiple gene knockouts are often employed in studies of microbial physiology and genetics. However, the selective markers that confer antibiotic resistance are generally limited, so it is necessary to remove these resistance genes before the next round of using, which is time consuming and labor intensive. Here, we created a universal circular gene knockout system for both the gram-negative bacterial Burkholderiales strain DSM 7029 and the gram-positive bacterial Mycobacterium smegmatis mc2 155, by combining the homologous recombination with multiple serine integrase-meditated site-specific recombination systems. In this system, a resistance gene and an integrase gene were constructed within the two attachment sites corresponding to a second, different integrase to form a cassette for gene disruption, which could be easily removed by the second integrase during the subsequent round of gene knockout. The sacB gene was also employed for negative selection. As the integrase-mediated deletion of the resistance/integrase gene cassette was highly efficient and concurrent with the following knockout round, the cyclic use of three cassettes could achieve multiple gene knockout in a sequential manner. Following the modularity concept in synthetic biology, common components of the knockout plasmids were retained as BioBricks, accelerating the knockout plasmids construction process. The circular gene knockout system can also be used for multiple gene insertions and applied to other microorganisms.

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Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate

BioTechniques ◽

10.2144/05386st03 ◽

2005 ◽

Vol 38 (6) ◽

pp. 891-894 ◽

Cited By ~ 60

Author(s):

Yoshihiro Nakajima ◽

Takuma Kimura ◽

Kazunori Sugata ◽

Toshiteru Enomoto ◽

Atsushi Asakawa ◽

...

Keyword(s):

Assay System ◽

Luciferase Assay ◽

Gene Expressions ◽

Multiple Gene ◽

Single Substrate ◽

One Step

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Innate Immunity to Mycobacterium tuberculosis

Clinical Microbiology Reviews ◽

10.1128/cmr.15.2.294-309.2002 ◽

2002 ◽

Vol 15 (2) ◽

pp. 294-309 ◽

Cited By ~ 347

Author(s):

Reinout van Crevel ◽

Tom H. M. Ottenhoff ◽

Jos W. M. van der Meer

Keyword(s):

Innate Immunity ◽

Mycobacterium Tuberculosis ◽

Host Defense ◽

Crucial Role ◽

Defense Mechanisms ◽

Gene Knockout ◽

Cell Mediated Immunity ◽

Immune Recognition ◽

Humoral Factors ◽

Genetic Studies

SUMMARY The different manifestations of infection with Mycobacterium tuberculosis reflect the balance between the bacillus and host defense mechanisms. Traditionally, protective immunity to tuberculosis has been ascribed to T-cell-mediated immunity, with CD4+ T cells playing a crucial role. Recent immunological and genetic studies support the long-standing notion that innate immunity is also relevant in tuberculosis. In this review, emphasis is on these natural, innate host defense mechanisms, referring to experimental data (e.g., studies in gene knockout mice) and epidemiological, immunological, and genetic studies in human tuberculosis. The first step in the innate host defense is cellular uptake of M. tuberculosis, which involves different cellular receptors and humoral factors. Toll-like receptors seem to play a crucial role in immune recognition of M. tuberculosis, which is the next step. The subsequent inflammatory response is regulated by production of pro- and anti-inflammatory cytokines and chemokines. Different natural effector mechanisms for killing of M. tuberculosis have now been identified. Finally, the innate host response is necessary for induction of adaptive immunity to M. tuberculosis. These basic mechanisms augment our understanding of disease pathogenesis and clinical course and will be of help in designing adjunctive treatment strategies.

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