Biology subjects

Hamadache, S.

Publications and source records attributed to Hamadache, S..

3 recordsLinked to original sources

bioRxiv2025

Engineering Conjugative Plasmids for Inducible Horizontal DNA Transfer

Rapidly developing microbial resistance to existing antimicrobials poses a growing threat to public health and global food security. Current chemical-based treatments target cells by inhibiting growth or metabolic function, but their effectiveness is diminishing. To address the growing antimicrobial resistance crisis, there is an urgent need for innovative therapies. Conjugative plasmids, a natural mechanism of horizontal gene transfer in bacteria, have been repurposed to deliver toxic genetic cargo to recipient cells, showing promise as next-generation antimicrobial agents. However, the ecological risks posed by unintended gene transfer require robust biocontainment strategies. In this study, we developed inducible conjugative plasmids to solve these challenges. Utilizing an arabinose-inducible promoter, we evaluated 13 plasmids with single essential gene deletions, identifying trbC and trbF as strong candidates for stringent regulation. These plasmids demonstrated inducibility in both cis and trans configurations, with induction resulting in up to a 5-log increase in conjugation efficiency compared to uninduced conditions. Although challenges such as reduced conjugation efficiency and promoter leakiness persist, this work establishes a foundation for the controlled transfer of plasmids, paving the way for safer and more effective antimicrobial technologies.

synthetic biology↗

bioRxiv2024

Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd

Haemophilus influenzae is a bacterial species of interest for its medical relevance and utility as a model system. Despite its role in several landmark molecular and synthetic biology studies, H. influenzae remains underexplored as a potential chassis organism. The limited availability of reliable and convenient transformation methods and genetic tools for H. influenzae are obstacles to this end. However, a strain of H. influenzae Rd KW20 lacking the type II restriction endonucleases HindII and HindIII has previously been developed. Here, we show that this strain is more readily transformable by electroporation than wild-type Rd KW20. We also developed a series of multi-host plasmids carrying antibiotic selection and fluorescent visual markers based on the pSU20 vector. The availability of H. influenzae {Delta}HindII/III, paired with the electroporation method and plasmids presented here, will promote the exploration of H. influenzae as a host organism for synthetic biology applications.

synthetic biology↗

bioRxiv2019

Rapid method for generating designer algal mitochondrial genomes

With synthetic biology, we can turn algae into bio-factories that produce high-value molecules (e.g. medicines or biofuels) or tackle global challenges (e.g. malnutrition and climate change). This realization has provoked rapid progress towards the creation of genetic tools for multiple algal species, notably Phaeodactylum tricornutum. The power of synthetic biology to generate more useful or productive organisms is contingent on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants. However, it is still relatively expensive to synthesize DNA, and delivering large DNA (>50 kbp) to eukaryotic cellular compartments remains challenging. In this study, we establish a robust system for building algal mitochondrial genomes as a practical alternative to DNA synthesis. Our approach permits an inexpensive and rapid generation of mitochondrial derivatives designed for testing targeted DNA delivery. First, we cloned the mitochondrial genome of P. tricornutum into the eukaryotic host organism S. cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we analyzed the cloned genomes by multiplex PCR, and correct genomes were transferred to prokaryotic host E. coli. Then these genomes were again analyzed by multiplex PCR, followed by diagnostic digest and complete-plasmid sequencing to evaluate the fidelity of each cloning method. Finally, we assessed the burden on eukaryotic and prokaryotic hosts to propagate the cloned genomes. We conclude that our system can reliably generate variants for genome-level engineering of algal mitochondria. HIGHLIGHTSO_LITAR-cloned mitochondrial genome of P. tricornutum in yeast C_LIO_LIDeveloped PCR-based cloning method to create designer algal mitochondrial genomes C_LIO_LIStably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts C_LI

synthetic biology↗