36 results found
Kelly CL, Harris AWK, Steel H, et al., 2018, Synthetic negative feedback circuits using engineered small RNAs., Nucleic Acids Res
Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs). In this work, we modelled, built and validated two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal, steep input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA, which inhibits translation of the mRNA encoding the output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.
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.
Little GT, Willson BJ, Heap JT, et al., 2018, The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools., Biotechnol J
The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The developed DNA transfer procedure allowed demonstration in NCIMB 14988 of the forward and reverse genetic techniques of transposon mutagenesis and gene knockout, respectively. The latter is accomplished through the successful deployment of both group II intron retargeting (ClosTron) and allelic exchange. In addition to gene inactivation, the developed allelic exchange procedure is used to create point mutations in the chromosome, allowing for the effect of amino acid changes in enzymes involved in primary metabolism to be characterized. ClosTron mediated disruption of the currently unannotated non-coding region between genes LF65_05915 and LF65_05920 is found to result in a non-sporulating phenotype.
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 Pthlwas 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 Pthlwas 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.
Taylor G, Mordaka P, Heap J, 2018, Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering.
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
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
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
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
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
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
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
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
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
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
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
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, ISSN: 0021-9258
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
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
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
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
Bradshaw M, Marshall KM, Heap JT, et al., 2010, Construction of a Nontoxigenic Clostridium botulinum Strain for Food Challenge Studies, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 76, Pages: 387-393, ISSN: 0099-2240
Burns DA, Heap JT, Minton NP, 2010, SleC Is Essential for Germination of Clostridium difficile Spores in Nutrient-Rich Medium Supplemented with the Bile Salt Taurocholate, JOURNAL OF BACTERIOLOGY, Vol: 192, Pages: 657-664, ISSN: 0021-9193
Burns DA, Heap JT, Minton NP, 2010, Clostridium difficile spore germination: an update, RESEARCH IN MICROBIOLOGY, Vol: 161, Pages: 730-734, ISSN: 0923-2508
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
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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