Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Antimicrobial resistance (AMR) is threatening the lives of millions worldwide. Antibiotics which once saved countless lives, are now failing, ushering in vaccines development as a current global imperative. Conjugate vaccines produced either by chemical synthesis or biologically in Escherichia coli cells, have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccines assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel method that overcomes the limitations of the current conjugate vaccine design method(s). We demonstrate at least 2-fold increase in glycoconjugate yield via MAGIC when compared to conventional bioconjugate method(s). Furthermore, the modularity of the MAGIC platform also allowed us to perform glycoengineering in genetically intractable bacterial species other than E. coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against bacteria, especially AMR pathogens, and could be applied for biopreparedness.

Scaling between DNA and cell size governs bacterial growth homeostasis and resource allocation

Bacteria maintain a stable cell size and a certain DNA content through proliferation as described by classic growth laws. How cells behave when this inherent scaling is broken, however, has rarely been interrogated. Here we engineered Escherichia coli cells with extremely low DNA contents using a tunable synthetic tool CRISPRori that temporarily inhibited chromosome replication initiation. A detailed mechanistic model coupling DNA replication, cell growth, and division revealed a fundamental DNA-centric growth law, which was validated by two observations. First, lineage dynamics were robust to large CRISPRori perturbations with division cycles rapidly restoring through a timer mechanism rather than the adder rule. Second, cellular growth transitioned into a linear regime at low DNA-cytoplasm ratios. Experiments and theory showed that in this regime, cellular resource was redirected to plasmid-borne gene expression. Together with the ability of CRISPRori to bi-directionally modulate plasmid copy numbers, these findings suggest a novel strategy for bio-production enhancement.

CONSTRUCTING A NOVEL PLASMID TO INCREASE PURITY EFFECTIVENESS FOR RECOMBINANT PROTEINS EXPRESSED IN ESCHERICHIA COLI CELLS

Nowadays, requirement of supply for recombinant proteins has increased in several fields such as food technology, medical pharmacy, clinical diagnose or environment treatment. The recombinant proteins have become the commercialized products and of that yielded with increasing in a large number per year. Besides, supposing that on extending of the his-tag of the pET111a plasmid may be facilitate for removing his-tag and more effective in protein purification. In this study, nine nucleotides (GCGGCGGCG) coding three alanine residues were added to positions followed hexa-histidine tag (his-tag) on a pET11a plasmid construction. The SDS-PAGE result of each recombinant protein contained the long-modified tag after purification process almost only exhibited single band on gel. Based on alike observed consequences for three recombinant proteins already refined the purity effectiveness reach to upper 98% in the total of existing proteins inside the solution. Hence, the novel pET11a plasmid construction could become an effective plasmid for the aim of harvesting high-purified recombinant proteins.

Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment

Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism’s phenotypic survival to antibiotics; however, this hypothesis remains to be tested.