Biology subjects

Heap, J. T.

Publications and source records attributed to Heap, J. T..

3 recordsLinked to original sources

bioRxiv2018

Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering.

DNA assembly allows individual DNA constructs or designed mixtures 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 minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in E. coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC=\"FIGDIR/small/361626_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (45K):\norg.highwire.dtl.DTLVardef@1cdd32eorg.highwire.dtl.DTLVardef@1e394cforg.highwire.dtl.DTLVardef@3ec8f5org.highwire.dtl.DTLVardef@1fb72e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2017

Stringency of synthetic promoter sequences in Clostridium revealed and circumvented by tuning promoter library mutation rates

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 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.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC=\"FIGDIR/small/216853_ufig1.gif\" ALT=\"Figure 1000\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@52f2borg.highwire.dtl.DTLVardef@5be460org.highwire.dtl.DTLVardef@ee8b05org.highwire.dtl.DTLVardef@e92801_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2017

Synthetic negative feedback circuits using engineered small RNAs

Negative feedback is known to endow biological and man-made systems with robust performance in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules which can inhibit translation of target messenger RNAs (mRNAs). In this paper, we designed, modelled and built 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 input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA which negatively regulates the translation of the mRNA encoding this 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.

synthetic biology↗