A Synthetic Plasmid Toolkit for Shewanella oneidensis MR-1

Abstract

Shewanella oneidensis MR-1 is a platform microorganism for understanding extracellular electron transfer (EET) with a fully sequenced and annotated genome. In comparison to other model microorganisms such as Escherichia coli, the available plasmid parts (such as promoters and replicons) are not sufficient to conveniently and quickly fine-tune the expression of multiple genes in S. oneidensis MR-1. Here, we constructed and characterized a plasmid toolkit that contains a set of expression vectors with a combination of promoters, replicons, antibiotic resistance genes, and an RK2 origin of transfer (oriT) cassette, in which each element can be easily changed by fixed restriction enzyme sites. The expression cassette is also compatible with BioBrick synthetic biology standards. Using green fluorescent protein (GFP) as a reporter, we tested and quantified the strength of promoters. The copy number of different replicons was also measured by real-time quantitative PCR. We further transformed two compatible plasmids with different antibiotic resistance genes into the recombinant S. oneidensis MR-1, enabling control over the expression of two different fluorescent proteins. This plasmid toolkit was further used for overexpression of the MtrCAB porin-c-type cytochrome complex in the S. oneidensis ΔmtrA strain. Tungsten trioxide (WO3) reduction and microbial fuel cell (MFC) assays revealed that the EET efficiency was improved most significantly when MtrCAB was expressed at a moderate level, thus demonstrating the utility of the plasmid toolkit in the EET regulation in S. oneidensis. The plasmid toolkit developed in this study is useful for rapid and convenient fine-tuning of gene expression and enhances the ability to genetically manipulate S. oneidensis MR-1.

Developing a population-state decision system for intelligently reprogramming extracellular electron transfer in Shewanella oneidensis

“…A further precise tuning of the PSD system requires genetic regulatory elements of different strengths. Although molecular tools have been advanced recently in S. oneidensis (33,34), the well-refined expression elements, for example, promoters and ribosome-binding sequence, are still limited in S. oneidensis, which restricts the construction of a set of PSD variants covering wide-ranging output strengths. Therefore, it is essential to explore and construct a kit of a plentiful number of regulatory elements in S. oneidensis and other EAB species, and utilize them in combination to modulate and customize the PSD system outputs for different purposes.…”

Synthetic biology approaches towards the recycling of metals from the environment

“…Pseudomonas for example has a number of characterised induction systems and a method of recombineering [ 84 ]. While a modular plasmid toolkit, based on the BioBrick assembly method, was recently reported for use with Shewanella and demonstrated in the reduction in tungsten [ 85 ].…”

Engineering controllable biofilms for biotechnological applications

“…Synthetic biology holds considerable promise for controlling biofilms by improving and expanding existing molecular tools, introducing novel functions to the system, modulating gene regulation and protein expression (Fu, 2006; Brenner et al ., 2008). However, the shortage of well‐defined gene expression tools in a diverse range of bacteria restricts the scope of synthetic biology applications in potential bacteria relevant to biotechnological applications (Cao et al ., 2019). Future efforts should focus on developing a systematic toolkit to expand our ability to genetically manipulate a diverse range of non‐model bacteria that have been shown to play important roles in biofilm‐mediated bioprocesses.…”