37 results found
Taylor GM, Mordaka PM, Heap JT, 2018, Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering., Nucleic Acids Res
DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable 'scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.
Little GT, Willson BJ, Heap JT, et al., 2018, The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools, BIOTECHNOLOGY JOURNAL, Vol: 13, ISSN: 1860-6768
Kelly CL, Harris AWK, Steel H, et al., 2018, Synthetic negative feedback circuits using engineered small RNAs, NUCLEIC ACIDS RESEARCH, Vol: 46, Pages: 9875-9889, ISSN: 0305-1048
Kelly CL, Taylor GM, Hitchcock A, et al., 2018, A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria., ACS Synth Biol, Vol: 7, Pages: 1056-1066
Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.
Taylor G, Mordaka P, Heap J, 2018, Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering.
Mordaka PM, Heap JT, 2018, Stringency of Synthetic Promoter Sequences in Clostridium Revealed and Circumvented by Tuning Promoter Library Mutation Rates, ACS Synthetic Biology, Vol: 7, Pages: 672-681
© 2018 American Chemical Society. Collections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially relevant Clostridium spp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria like Clostridium. We directly compared oxygen-independent reporters of different types in Clostridium acetobutylicum and found that glucuronidase (GusA) from E. coli performed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (Pthl) except for the consensus -35 and -10 elements. In each synthetic promoter, the chance of each degenerate position matching Pthl was 25%. Surprisingly, none of the tested synthetic promoters from this library were functional in C. acetobutylicum, even though they functioned as expected in E. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching Pthl was 79% or 58%, instead of 25% as before. Synthetic promoters from these "stringent" libraries functioned well in C. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly related species Clostridium sporogenes, and allowed predictable metabolic engineering of C. acetobutylicum for acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected "stringency" of promoter sequences in Clostridium, not reported previously.
Sellés Vidal L, Kelly CL, Mordaka PM, et al., 2018, Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application., Biochim Biophys Acta Proteins Proteom, Vol: 1866, Pages: 327-347, ISSN: 1570-9639
NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P)+. NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.
Jain S, Smyth D, O'Hagan BMG, et al., 2017, Inactivation of the dnaK gene in Clostridium difficile 630 Delta erm yields a temperature-sensitive phenotype and increases biofilm-forming ability, SCIENTIFIC REPORTS, Vol: 7, ISSN: 2045-2322
Miari VF, Solanki P, Hieba Y, et al., 2017, In Vitro Susceptibility to Closthioamide among Clinical and Reference Strains of Neisseria gonorrhoeae, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol: 61, ISSN: 0066-4804
Walker DJF, Heap JT, Winzer K, et al., 2016, A genetic assay for gene essentiality in Clostridium, ANAEROBE, Vol: 42, Pages: 40-43, ISSN: 1075-9964
Kelly CL, Liu Z, Yoshihara A, et al., 2016, Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter., ACS Synth Biol, Vol: 5, Pages: 1136-1145
External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.
Liu Z, Yoshihara A, Kelly C, et al., 2016, 6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 22, Pages: 12557-12565, ISSN: 0947-6539
Ehsaan M, Kuit W, Zhang Y, et al., 2016, Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824, Biotechnology for Biofuels, Vol: 9, Pages: 4-4, ISSN: 1754-6834
BACKGROUND: Clostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum. RESULTS: Both systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1). CONCLUSIONS: The codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic eff
Heap JT, Theys J, Ehsaan M, et al., 2014, Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo, ONCOTARGET, Vol: 5, Pages: 1761-1769, ISSN: 1949-2553
Kovacs K, Willson BJ, Schwarz K, et al., 2013, Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824, BIOTECHNOLOGY FOR BIOFUELS, Vol: 6, ISSN: 1754-6834
Jabbari S, Steiner E, Heap JT, et al., 2013, The putative influence of the agr operon upon survival mechanisms used by Clostridium acetobutylicum, MATHEMATICAL BIOSCIENCES, Vol: 243, Pages: 223-239, ISSN: 0025-5564
Zhang Z, Korkeala H, Dahlsten E, et al., 2013, Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502, PLOS PATHOGENS, Vol: 9, ISSN: 1553-7366
Lindstrom M, Dahlsten E, Soderholm H, et al., 2012, Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 78, Pages: 5466-5470, ISSN: 0099-2240
Cartman ST, Kelly ML, Heeg D, et al., 2012, Precise Manipulation of the Clostridium difficile Chromosome Reveals a Lack of Association between the tcdC Genotype and Toxin Production, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 78, Pages: 4683-4690, ISSN: 0099-2240
Saad NY, Schiel B, Braye M, et al., 2012, Riboswitch (T-box)-mediated Control of tRNA-dependent Amidation in Clostridium acetobutylicum Rationalizes Gene and Pathway Redundancy for Asparagine and Asparaginyl-tRNA(Asn) Synthesis, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 20382-20394
Heap JT, Ehsaan M, Cooksley CM, et al., 2012, Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker, NUCLEIC ACIDS RESEARCH, Vol: 40, ISSN: 0305-1048
Selby K, Lindstrom M, Somervuo P, et al., 2011, Important Role of Class I Heat Shock Genes hrcA and dnaK in the Heat Shock Response and the Response to pH and NaCl Stress of Group I Clostridium botulinum Strain ATCC 3502, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 77, Pages: 2823-2830, ISSN: 0099-2240
Steiner E, Dago AE, Young DI, et al., 2011, Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum, MOLECULAR MICROBIOLOGY, Vol: 80, Pages: 641-654, ISSN: 0950-382X
Soderholm H, Lindstrom M, Somervuo P, et al., 2011, cspB encodes a major cold shock protein in Clostridium botulinum ATCC 3502, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, Vol: 146, Pages: 23-30, ISSN: 0168-1605
Jabbari S, Heap JT, King JR, 2011, Mathematical Modelling of the Sporulation-Initiation Network in Bacillus Subtilis Revealing the Dual Role of the Putative Quorum-Sensing Signal Molecule PhrA, BULLETIN OF MATHEMATICAL BIOLOGY, Vol: 73, Pages: 181-211, ISSN: 0092-8240
Burns DA, Heap JT, Minton NP, 2010, The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type, ANAEROBE, Vol: 16, Pages: 618-622, ISSN: 1075-9964
Burns DA, Heap JT, Minton NP, 2010, Clostridium difficile spore germination: an update, RESEARCH IN MICROBIOLOGY, Vol: 161, Pages: 730-734, ISSN: 0923-2508
Kuehne SA, Cartman ST, Heap JT, et al., 2010, The role of toxin A and toxin B in Clostridium difficile infection, NATURE, Vol: 467, Pages: 711-U97, ISSN: 0028-0836
Cartman ST, Heap JT, Kuehne SA, et al., 2010, The emergence of 'hypervirulence' in Clostridium difficile, INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, Vol: 300, Pages: 387-395, ISSN: 1438-4221
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