THE ABCS OF GENE CLONING

By OLIVIA ROBINSON

1 THE FUNCTION AND ORGANIZATION OF PLASMIDS 2 CHOOSING A CLONING VECTOR 3 ESCHERICHIA COLI HOST STRAINS 4 CHEMICAL TRANSFORMATION OF E COLI 5 ELECTROPORATION OF E COLI 6 DNA TRANSFER BY BACTERIAL CONJUGATION 7 COSMID PACKAGING AND INFECTION OF E COLI 8 ISOLATION OF PLASMIDS FROM E COLI BY ALKALINE LYSIS 9 ISOLATION OF PLASMIDS FROM E COLI BY BOILING LYSIS 10 HIGH-PURITY PLASMID ISOLATION USING SILICA OXIDE 11 HIGH-THROUGHPUT PLASMID EXTRACTION USING MICROTITER PLATES 12 ISOLATION OF COSMID AND BAC DNA FROM E COLI 13 PREPARATION OF SINGLE-STRANDED DNA FROM PHAGEMID VECTORS 14 USING DESKTOP CLONING SOFTWARE TO PLAN, TRACK, AND EVALUATE CLONING PROJECTS 15 CLONING IN PLASMID VECTORS 16 EXTRACTION OF DNA FROM AGAROSE GELS 17 CLONING PCR PRODUCTS WITH T-VECTORS CONTENTS 18 CONSTRUCTION OF GENOMIC LIBRARIES IN ?-VECTORS 19 RAPID SCREENING OF RECOMBINANT PLASMIDS 20 RESTRICTION ANALYSIS OF RECOMBINANT PLASMIDS 21 SCREENING RECOMBINANT DNA LIBRARIES 22 SEQUENCING USING FLUORESCENT-LABELED NUCLEOTIDES 23 SITE-DIRECTED MUTAGENESIS USING THE MEGAPRIMER METHOD 24 SITE-DIRECTED MUTAGENESIS BY INVERSE PCR 25 CREATING NESTED DNA DELETIONS USING EXONUCLEASE III 26 TRANSPOSON AND TRANSPOSOME MUTAGENESIS OF PLASMIDS, COSMIDS, AND BACS 27 IN VITRO TRANSCRIPTION AND TRANSLATION 28 VECTORS FOR THE EXPRESSION OF RECOMBINANT PROTEINS IN E COLI 29 EXPRESSION OF RECOMBINANT PROTEINS FROM LAC PROMOTERS 30 PLASMID-BASED REPORTER GENES: ASSAYS FOR ?-GALACTOSIDASE AND ALKALINE PHOSPHATASE ACTIVITIES 31 PLASMID-BASED REPORTER GENES: ASSAYS FOR GREEN FLUORESCENT PROTEIN THE FUNCTION AND ORGANIZATION OF PLASMIDS 1 INTRODUCTION IN 1952, JOSHUA LEDERBERG COINED THE TERM PLASMID TO DESCRIBE ANY BACTERIAL GENETIC ELEMENT THAT EXISTS IN AN EXTRACHROMOSOMAL STATE FOR AT LEAST PART OF ITS REPLICATION CYCLE (1) AS THIS DESCRIPTION INCLUDED BACTERIAL VIRUSES, THE DEFINITION OF WHAT CONSTI- TUTES A PLASMID WAS SUBSEQUENTLY REFINED TO DESCRIBE EXCLUSIVELY OR PREDOMINANTLY EXTRACHROMOSOMAL GENETIC ELEMENTS THAT REPLICATE AUTONOMOUSLY PLASMIDS ARE NOW KNOWN TO BE PRESENT IN MOST SPECIES OF EUBACTERIA THAT HAVE BEEN EXAMINED, AS WELL AS IN ARCHAEA AND LOWER EUKARYA (2) ALTHOUGH MOST OF THE GENETIC MATERIAL THAT DIRECTS THE STRUCTURE AND FUNCTION OF A BACTERIAL CELL IS CONTAINED WITHIN THE CHROMOSOME, PLASMIDS CONTRIBUTE SIGNIFICANTLY TO BACTERIAL GENETIC DIVERSITY AND PLASTICITY BY ENCODING FUNCTIONS THAT MIGHT NOT BE SPECIFIED BY THE CHROMOSOME (3) (SEE SUBHEADING 3) FOR EXAMPLE, ANTIBIOTIC RESIS- TANCE GENES ARE OFTEN PLASMID-ENCODED, WHICH ALLOWS THE BACTERIUM TO PERSIST IN AN ANTIBIOTIC-CONTAINING ENVIRONMENT, THEREBY PROVIDING THE BACTERIUM WITH A COMPETI- TIVE ADVANTAGE OVER ANTIBIOTIC-SENSITIVE SPECIES UNDER LABORATORY CONDITIONS, PLASMIDS ARE GENERALLY NOT ESSENTIAL FOR THE SURVIVAL OF THE HOST BACTERIUM AND THEY HAVE SERVED AS INVALUABLE MODEL SYSTEMS FOR THE STUDY OF PROCESSES SUCH AS DNA REPLICATION, SEGREGATION, CONJUGATION, AND EVOLUTION (3) MORE- OVER, EVER SINCE THEIR UTILITY WAS EVINCED BY THE FIRST GENE-CLONING EXPERIMENTS IN THE EARLY 1970S, PLASMIDS HAVE BEEN PIVOTAL TO MODERN RECOMBINANT DNA TECHNOLOGY AS GENE-CLONING AND GENE-EXPRESSION VEHICLES, AMONG OTHER USES (4,5) 2 BASIC PLASMID CHARACTERISTICS 2 1 SIZE AND COPY NUMBER NATURALLY OCCURRING PLASMIDS VARY GREATLY IN THEIR PHYSICAL PROPERTIES, A FEW EXAMPLES OF WHICH ARE SHOWN IN TABLE 1 THEY RANGE IN SIZE FROM <2-KILOBASE PAIR (KBP) PLAS- MIDS, WHICH CAN BE CONSIDERED TO BE ELEMENTS SIMPLY CAPABLE OF REPLICATION, TO HAYES MEGAPLASMIDS THAT ARE MANY HUNDREDS OF KILOBASE PAIRS IN SIZE AT THE UPPER END OF THIS SCALE, THE DISTINCTION BETWEEN A MEGAPLASMID AND A MINICHROMOSOME CAN BECOME OBSCURE SOME BACTERIAL SPECIES SIMULTANEOUSLY HARBOR MULTIPLE DIFFERENT PLASMIDS THAT CAN CONTRIBUTE SIGNIFICANTLY TO THE OVERALL GENOME SIZE OF THE HOST BACTERIUM (SEE FIG 1) (6,13) AS AN EXAMPLE, THE SYMBIOTIC SOIL BACTERIUM SINORHIZOBIUM MELILOTI HAS THREE REPLICONS (3 65, 1 68, AND 1 35 MEGABASE PAIRS [MBP]) IN ADDITION TO ITS CHROMOSOME (6 69 MBP) (8) THE SMALLEST MEGAPLASMID, PSYMA, CAN BE CURED FROM THE HOST BACTE- RIUM UNDER LABORATORY CONDITIONS BUT PROVIDES NODULATION AND NITROGEN-FIXATION FUNC- TIONS THAT ARE IMPORTANT FOR THE SYMBIOTIC INTERACTION OF THE BACTERIUM AND ITS PLANT HOST TABLE LINEAR AARCHAEA FIG 1 PLASMID COMPLEMENT OF A MULTIPLASMID-CONTAINING STRAIN OF LACTOCOCCUS LACTIS ANA- LYZED BY AGAROSE GEL ELECTROPHORESIS THE APPROXIMATE SIZES OF THE PLASMIDS ARE INDICATED (KBP) THE ABCS OF GENE CLONING