Towards a Yersinia pestis lipid A recreated in an Escherichia coli scaffold genome

TL;DR

Scientists tried to recreate a specific bacterial component from Yersinia pestis in Escherichia coli but did not achieve the expected result.

Contribution

The study demonstrates the insertion of Yersinia pestis lipid A biosynthesis genes into E. coli and evaluates their expression and functionality.

Findings

Three Y. pestis lipid A biosynthesis genes were successfully inserted into the E. coli genome.

Expression of the inserted genes was confirmed via RT-PCR.

The engineered E. coli did not produce the expected Y. pestis lipid A structure.

Abstract

Synthetic biology and genome engineering capabilities have facilitated the utilization of bacteria for a myriad of applications, ranging from medical treatments to biomanufacturing of complex molecules. The bacterial outer membrane, specifically the lipopolysaccharide (LPS), plays an integral role in the physiology, pathogenesis, and serves as a main target of existing detection assays for Gram-negative bacteria. Here we use CRISPR/Cas9 recombineering to insert Yersinia pestis lipid A biosynthesis genes into the genome of an Escherichia coli strain expressing the lipid IVa subunit. We successfully inserted three genes: kdsD, lpxM, and lpxP into the E. coli genome and demonstrated their expression via reverse transcription PCR (RT-PCR). Despite observing expression of these genes, analytical characterization of the engineered strain’s lipid A structure via MALDI-TOF mass spectrometry indicated that the Y. pestis lipid A was not recapitulated in the E. coli background. As synthetic biology and genome engineering technologies advance, novel applications and utilities for the detection and treatments of dangerous pathogens like Yersinia pestis will continue to be developed.

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