A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.
New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans. These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allows transformation frequencies of as high as 2 x 10(8) transformants per microgram of DNA per 10(9) cells or approximately 20% of the recipient population. During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined. The implications of these findings for the future of methanoarchaeal research are also discussed.
Metcalf
W W
WW
Department of Microbiology, University of Illinois, Urbana 61801, USA. bill_metcalf@qms1.life.uiuc.edu
Zhang
J K
JK
Apolinario
E
E
Sowers
K R
KR
Wolfe
R S
RS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004261:DNA Replication; D004337:Drug Carriers; D004926:Escherichia coli; D008081:Liposomes; D017020:Methanosarcina / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping
D017931:DNA Primers; D004337:Drug Carriers; D008081:Liposomes