Multiple Cloning Site

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Rudi Beyaert - One of the best experts on this subject based on the ideXlab platform.

Jens Staal - One of the best experts on this subject based on the ideXlab platform.

Patrice Courvalin - One of the best experts on this subject based on the ideXlab platform.

  • shuttle vectors containing a Multiple Cloning Site and a laczα gene for conjugal transfer of dna from escherichia coli to gram positive bacteria
    Gene, 1991
    Co-Authors: Patrick Trieucuot, C Carlier, Claire Poyartsalmeron, Patrice Courvalin
    Abstract:

    Abstract The mobilizable shuttle Cloning vectors, pATIS and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAMβ1; (ii) an erythromycin-resistance-encoding gene expressed in Gram− and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the Multiple Cloning Site and the lacZα reporter gene of pUCIS (pATIS) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique Cloning Sites that allow screening of derivatives containing DNA inserts by α-complementation in Escherichia coli carrying the lacZAM15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pATIS, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10−6 to 10−9. The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.

Wouter De Schamphelaire - One of the best experts on this subject based on the ideXlab platform.

D. J. Leblanc - One of the best experts on this subject based on the ideXlab platform.

  • Improved electroporation and Cloning vector system for gram-positive bacteria
    Applied and Environmental Microbiology, 1991
    Co-Authors: G. M. Dunny, L. N. Lee, D. J. Leblanc
    Abstract:

    A protocol for transformation of intact Enterococcus faecalis cells by electroporation was developed through a systematic examination of the effects of changes in various parameters, including (i) growth conditions; (ii) composition of the electroporation solution; (iii) electroporation conditions, such as field strength and resistance; (iv) size, concentration, and purity of DNA used for transformation; and (v) conditions used to select for transformants. Key features of this protocol include the use of exponential-phase cells grown in inhibitory concentrations of glycine and the use of an acidic sucrose electroporation solution. Frequencies of greater than 2 x 10(5) transformants per microgram of plasmid DNA were obtained for E. faecalis cells, whereas various strains of streptococci and Bacillus anthracis were transformed at frequencies of 10(3) to 10(4) transformants per microgram of plasmid DNA with the same protocol. A novel Escherichia coli-Streptococcus and Enterococcus shuttle Cloning vector, pDL276, was constructed for use in conjunction with the electroporation system. This vector features a Multiple Cloning Site region flanked by E. coli transcription termination sequences, a relatively small size (less than 7 kb), and a kanamycin resistance determinant expressed in both gram-positive and gram-negative hosts. Various enterococcal and streptococcal DNA sequences were cloned in E. coli (including sequences that could not be cloned on other vectors) and were returned to the original host by electroporation. The vector and electroporation system was also used to clone directly into E. faecalis.