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Vectors with the fd replicon for in vivo cloning and analysis of genes
M Osuský1, S Stuchlík, M Zámocký
1Department of Biochemistry, Comenius University, Bratislava, Slovakia.
Abstract:
We have constructed two new mini-Mu derivatives, pMRfP and pBEf, that combine the properties of known mini-Mu vectors and the advantages of the replication origin (orifd) of filamentous phage fd. Mini-Mu pMRfP consists of the left (850 bp) and the right (216 bp) ends of the Mu genome, orifd, packaging signal of fd, and the gene conferring resistance to chloramphenicol. The second mini-Mu, termed pBEf, carries the left end of Mu (1001 bp), which contains the so-called internal activation sequence (enhancer of transposition), required for a higher frequency of transposition, the right end (116 bp) and the gene conferring resistance to kanamycin. These new mini-Mu vectors are suitable for in vivo cloning with the ability of single-stranded DNA preparation using one of the helper phages (M13K07, rv1, IR1, R408) and with a large cloning capacity (the size of the cloned fragment can be up to 35 kb). They can also be used as the hoppers (a transposable ori that can be turned on or off depending on the presence of the fd gene 2 product). Thus, these mini-Mu derivatives can be employed as vectors for in vivo cloning, and as regulated transposons or mobile replicons.
Insights
Two novel mini-Mu derivatives, pMRfP and pBEf, were created for efficient in vivo cloning and as regulated transposons. These vectors leverage filamentous phage fd replication origins for enhanced functionality and large insert capacity.
Area of Science:
- Molecular Biology
- Bacteriophage Genetics
Background:
- Mini-Mu elements are versatile tools for genetic manipulation.
- Filamentous phage fd replication origins offer unique advantages for vector design.
Purpose of the Study:
- To construct and characterize novel mini-Mu derivatives with enhanced cloning capabilities.
- To integrate the replication origin of filamentous phage fd into mini-Mu vectors.
Main Methods:
- Construction of two mini-Mu derivatives, pMRfP and pBEf, incorporating Mu genome ends and fd replication origins.
- Characterization of vector properties including cloning capacity and single-stranded DNA preparation.
Main Results:
- pMRfP and pBEf combine Mu ends, fd replication origin (orifd), and antibiotic resistance genes (chloramphenicol and kanamycin, respectively).
- Vectors support large insert cloning up to 35 kb and enable single-stranded DNA preparation with helper phages.
- Demonstrated utility as hoppers, functioning as regulated transposable origins.
Conclusions:
- The new mini-Mu derivatives offer significant advantages for in vivo cloning and genetic engineering.
- These vectors serve as versatile tools for applications requiring regulated transposition and mobile replicons.