Shuttle Vector pBRLP31-8 Multi-Host Replication
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Solution Overview
Problem
There is a need for novel shuttle vectors that can replicate in Lactobacillus prokaryotic cells and other non-Lactobacillus prokaryotic cells, such as Escherichia coli and Bacillus subtilis, to facilitate genetic manipulation and protein production across multiple host species.
Innovation Solution
A shuttle vector, pBRLP31-8, is constructed using the minimal cryptic plasmid pLP31-8 from Lactobacillus and the replication region of E. coli plasmid pBR322, incorporating an E. coli plasmid replicon, a Lactobacillus plasmid replicon, a multiple cloning site, and a selectable marker like the chloramphenicol resistance gene, enabling replication in Lactobacillus plantarum, L. rhamnosus, W. cibaria, and B. subtilis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a plasmid is designed to replicate in multiple phylogenetically different species, then the versatility and host range are improved, but the plasmid structure becomes more complex requiring multiple replicon genes from different species
Solution Approach 1:
The patent combines the replicon gene from E. coli plasmid pACYC177 and the replicon gene from Enterococcus faecalis plasmid pAMĪ±1 into a single shuttle vector pHY300PLK. This merging of replicon elements from different species enables the plasmid to replicate in both E. coli and B. subtilis hosts, directly resolving the contradiction by achieving multi-host compatibility through structural integration of multiple replicon systems
Solution Approach 2:
The shuttle vector pHY300PLK is designed with dual replicon capability, allowing it to function as a replication origin in two different bacterial species (E. coli and B. subtilis). This multi-functional design enables a single plasmid structure to serve multiple host systems, improving versatility while maintaining a unified plasmid architecture rather than requiring separate plasmids for each host
2Ease of operation
If gene cloning is carried out in E. coli for ease of manipulation, then the ease of operation is improved, but the ability to directly express genes in target prokaryotic cells like Lactobacillus requires additional transformation steps
Solution Approach 1:
The shuttle vector incorporates both E. coli and Lactobacillus replicon elements, enabling it to replicate in both species. This allows researchers to perform gene cloning in E. coli (utilizing its ease of manipulation) and then directly transform the same plasmid into Lactobacillus for expression without requiring additional plasmid construction or adaptation steps, thereby reducing time loss while maintaining operational ease
Solution Approach 2:
The shuttle vector acts as an intermediary carrier that bridges E. coli and Lactobacillus systems. It facilitates the transfer of genetic material between these two species by maintaining replication capability in both hosts, allowing seamless transition from cloning in E. coli to expression in Lactobacillus without requiring intermediate plasmid modifications
Data Source
AI summary
A shuttle vector is provided which can be manipulated in various kinds of host cells, thereby providing a novel tool for the field of genetic engineering. Also provided are a prokaryotic host cell and a kit including said shuttle vector, so as to construct expression vectors which contain the target gene using the shuttle vector, thereby producing proteins in various host cells with one single vector.