Conjugative Plasmid Vectors Using Fructooligosaccharide Selection
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Solution Overview
Problem
Current genetic engineering technologies rely on antibiotic resistance markers for selecting bacteria carrying plasmid vectors, which is undesirable for research on antibacterial resistance mechanisms and in vivo applications, such as delivering vaccine antigens to the microbiota, as it poses risks of emerging antibiotic-resistant bacteria.
Innovation Solution
Development of conjugative plasmid vectors that utilize short-chain fructooligosaccharides as a selection marker, enabling bacteria to transfer the plasmid via conjugation without antibiotic resistance markers, allowing colonization and expression of genetic determinants in the gut microbiota.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If antibiotic resistance markers are used for selection, then bacteria carrying plasmid vectors can be easily selected, but there is a risk of emerging antibiotic-resistant bacteria
Solution Approach 1:
The patent extracts the selection marker function from antibiotic resistance genes and relocates it to a metabolic pathway (fructooligosaccharide utilization). The fos operon provides a selectable phenotype (ability to use scFOS as carbon source) without conferring antibiotic resistance, thereby eliminating the harmful side effect while preserving the selection function.
Solution Approach 2:
The patent changes the selection parameter from antibiotic resistance (binary presence/absence) to metabolic capability (growth on scFOS). This parameter change allows selection of transformed bacteria through their ability to metabolize fructooligosaccharides, providing an alternative selection mechanism that avoids antibiotic resistance issues.
2Productivity
If small plasmids with resistance markers are used, then transformation and propagation are efficient, but the vectors cannot transfer to other bacteria without helper plasmids
Solution Approach 1:
The patent merges the functions of small plasmids (efficient replication and selection) with conjugative plasmids (self-transfer capability) into a single vector system. The resulting conjugative plasmid maintains the advantages of both types: efficient transformation, selectable markers, and autonomous transfer to recipient bacteria without requiring helper plasmids.
Solution Approach 2:
The patent creates a multi-functional plasmid that simultaneously provides: (1) autonomous replication in bacterial hosts, (2) selectable phenotype through fos operon, (3) conjugation machinery for interbacterial transfer, and (4) expression of genetic determinants. This universal vector eliminates the need for separate helper plasmids.
3Measurement precision
If antibiotic resistance markers are used for selection, then transformed bacteria can be identified, but vectors without such markers are needed for in vivo applications
Solution Approach 1:
The patent introduces fructooligosaccharide metabolism as an intermediary selection system. The fos operon acts as a mediator that provides a selectable phenotype (growth on scFOS) without the harmful effects of antibiotic resistance. This intermediary mechanism enables both accurate selection and safe in vivo applications, including delivery of vaccine antigens to microbiota.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The plasmid vectors effectively colonize and express genetic information in the gut microbiota using scFOS as a carbon source, eliminating the need for antibiotic resistance markers and ensuring safe, efficient delivery of genetic information to intestinal bacteria.
Implementation Method 1
the fos operon, wherein the fos repressor is removed... a cluster of seven genes involved in the scFOS utilization pathway: fosK, fosY, fosGH2, fosX, fosGH1, fosT, and fosR
Implementation Method 2
conjugation is defined as a mechanism of horizontal gene transfer between bacterial cells involving cell-to-cell contact. During conjugation, the donor cell transfers a genetic element, most often a plasmid, to the recipient cell
Data Source
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AI summary
The invention relates to the generation of plasmid vectors comprising genes enabling broad-host range self-conjugation and a selection marker enabling use of a non-physiological carbon source. In some embodiments, the vectors are used in a method to colonize animals and humans intestinal flora, without the use of antimicrobials.