Biology subjects

Sikkema, A. P.

Publications and source records attributed to Sikkema, A. P..

3 recordsLinked to original sources

bioRxiv2026

Single amino acid substitution in DNA Polymerase I dramatically alters infection dynamics of bacteriophage T7

Viruses constitute a significant proportion of Earths genetic diversity, yet most remain uncharacterized beyond their sequences in viral metagenomes. Linking viral genotypes to phenotypes--especially enzyme function to phage infection dynamics--is challenging due to the lack of cultured virus-host systems. DNA polymerase I (PolA), essential for genome replication in [~]25% of dsDNA phages, provides an opportunity to explore these connections. In phage T7, residue 526 is critical for nucleotide incorporation, with previous in vitro evidence indicating impacts on enzyme efficiency and fidelity. Previous analyses identified three substitutions at this position (Tyr/Y, Phe/F, Leu/L) linked with deeply rooted viral PolA clades. Mutation impacts at residue 526 were tested in vitro and in vivo. The Y526F protein exhibited a 50% reduction in specific activity, and when introduced via High Complexity Golden Gate Assembly into T7 demonstrated a 53% decrease in burst size and significantly longer latent period compared to wild type. The Y526L protein exhibited a 97% decrease in activity, and the Y526L phage was incapable of completing its lifecycle. These findings confirm historical biochemical data, provide in vivo context for these mutations in the T7-E. coli system, and offer experimental support for genotype-to-phenotype associations in viral PolA, informing viral metagenomics studies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/726624v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@14395e5org.highwire.dtl.DTLVardef@261504org.highwire.dtl.DTLVardef@2dc1e4org.highwire.dtl.DTLVardef@147a7f_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Keown, R. (2026) https://BioRender.com/mhrmup3

microbiology↗

bioRxiv2023

One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse-genetics system

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of Infectious Bronchitis Virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

genetics↗

bioRxiv2020

Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by an order of magnitude and significantly improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300kDa) at 3.6 [A] resolution where density for individual side chains is clearly resolved.

biochemistry↗