coli S17-1 to other bacterial species quite often become modified by insertion of DNA from the donor host chromosome, presumably as a result of mobilization of DNA from the active oriT within the inserted RP4. In addition to these findings we here report that plasmids transferred from E. Furthermore, there are also reports describing generation of plasmid modifications of unknown nature in conjugation experiments involving E. Another demonstrated problem is that these strains not only mobilize oriT-carrying plasmids, but also their own chromosomal DNA to recipient strains at frequencies of 10 − 4 per donor cell. This may cause problems as Mu DNA may randomly mutate the recipient genome and/or the transferred plasmid.
coli strains S17-1 and SM10: They both contain an active bacteriophage Mu genome (within the tetracycline resistance gene of RP4) which has been shown to mobilize itself into recipient strains. In spite of their extensive use there are several problems associated with the E. A number of small and specialized oriT-containing vectors have been developed from the large RK2 plasmid (60 kb), but other types of plasmids containing oriT may also be conjugated by E. Conjugal transfer of plasmids based on this system requires the presence of an origin of transfer ( oriT) in the plasmid to be transferred, as well as the gene products of two separate tra-clusters which are provided in trans from the RP4 integrated in E. coli S17-1/SM10 contain a chromosomally integrated RP4 plasmid, which is essentially the same as the more studied broad-host-range self-transmissible IncP plasmid RK2. coli strain S17-1 and its analogue SM10 are heavily used as donor strains in such transfer procedures, which is reflected by a very high citation frequency (nearly 5000 as of October 2013) of the paper in which these strains are described. While the recipient-independency is an attractive feature, there also exist limitations due to the requirements of complex machinery and also due to protection systems in recipient cells, such as CRISPR and restriction-modification. The use of conjugation often solves these problems as the transfer system is mainly acting in a recipient-independent manner. Transformation of naked DNA is often inefficient, or sometimes even impossible, depending on the host of interest.
This becomes relevant for example during construction of large numbers of transposon insertion mutants or for transfer of metagenomic libraries in functional screening studies across species barriers. coli to alternative hosts at high frequencies. After the modifications have been made it might be necessary to transfer the constructs designed in E.
coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.ĭue to its well established genetics and good transformation competence Escherichia coli is the most frequently used host for manipulation of DNA via a variety of recombinant DNA technologies. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. We have observed that large plasmids were occasionally modified after conjugal transfer when using E.
coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. Plasmids bearing the RK2/RP4 origin of transfer ( oriT) are mostly mobilized using the E. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells.
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