Biology subjects

Hillson, N. J.

Publications and source records attributed to Hillson, N. J..

3 recordsLinked to original sources

bioRxiv2024

Bioinformatic prediction and high throughput in vivo screening to identify cis-regulatory elements for the development of algal synthetic promoters

Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, its development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2 or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high GFP expression. Further, we individually tested fourteen identified promoters, revealing substantial increases in GFP expression--up to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome dataset. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/585996v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1869cd4org.highwire.dtl.DTLVardef@a25d8dorg.highwire.dtl.DTLVardef@1abd96forg.highwire.dtl.DTLVardef@1501bc8_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on chip. The microfluidic device contains a 10 x 10 element array, each element containing sets of electrodes for two electric field actuated operations-electrowetting for merging droplets to mix reagents and electroporation for transformation. It can perform up to 100 genetic modifications in parallel, providing a scalable platform for generating the large number of engineered strains required for combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the systems capabilities through CRISPR-based engineering of two test cases-1) disruption of the function of enzyme galactokinase (galK) in E. coli and 2) targeted engineering of glutamine synthetase gene (glnA) and blue-pigment synthetase (bpsA) enzyme to improve indigoidine production in E. coli.

synthetic biology↗

bioRxiv2020

Characterization of an ECF56-family sigma factor from Streptomyces venezuelae reveals a highly conserved regulome

Bacteria often possess alternative sigma factors that initiate the transcription of specific genes under environmental stresses, the largest and most diverse group being the extracytoplasmic function (ECF) sigma factors. The regulation of ECF activity is crucial for ensuring the distinct transcription of stress responsive genes only occurs under the appropriate conditions. While most ECFs are comprised of only the core {sigma}2 and {sigma}4 regions, a unique form of ECF sigma factor regulation also contains a C-terminal extension bearing homology to the NTF2 superfamily of protein domains. While previous work has shown that this NTF2 domain can affect transcriptional activity in vivo in ECF41 and ECF42, its role in the newly classified ECF56 subgroup is unknown. In this work, we show that truncation of the C-terminus of the ECF56 sigma factor SVEN_4562 of Streptomyces venezuelae upregulates its activity in a hybrid assay. Through transcriptomics in S. venezuelae, we found that this truncated ECF56 sigma factor has a highly conserved promoter sequence in vivo. Bioinformatic assays illustrated that deep branches of the Actinobacteria phylum contained putative ECF56 promoter motifs identical to those found in the S. venezuelae ECF56 regulon. We validated these findings through ex situ hybrid assays illustrating that truncated ECF56 sigma factors from phylogenetically diverse Actinobacteria activate transcription from these promoters. Importantly, our work shows that the genetic infrastructure of the ECF56 family of sigma factors is highly conserved and performs important functions yet to be understood in Actinobacteria. ImportanceMost ECF sigma-factors rely on anti-sigma factor regulation; in contrast, the unique classes of ECF sigma-factors that contain a C-terminal extension are thought to respond directly to an environmental signal. Here we show that the cis-acting regulatory element of the ECF56 regulon is likely highly conserved in many Actinobacteria, with exact nucleotide level conservation over ~2 billion years of evolution. The high conservation of this genetic architecture, as well as a conserved gene content within the regulon, strongly point to a specialized and important role in Actinobacteria biology.

microbiology↗