Plasmids and Vectors Aims: Must be able to outline what plasmids and vectors are. Should be able to explain how plasmids and vectors are utilised in research. Could be able to outline the processes for gene cloning.

Bacterium: Agrobacterium tumefaciens Plasmid Vectors Plasmids are accessory chromosomes occurring naturally in bacteria. In nature, plasmids are usually transferred between closely related microbes by cell-to-cell contact (a process called conjugation). Simple chemical treatments can make mammalian cells, yeast cells and some bacterial cells that do not naturally transfer DNA, able to take up external DNA. Plant infected by bacterium with foreign gene Bacterium: Agrobacterium tumefaciens Plasmid Plasmid with foreign gene

Recombinant DNA Plasmid The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule of recombinant DNA. These combined techniques of using restriction enzymes and ligation are the basic tools of genetic engineering. DNA ligase G A T C Recombinant Plasmid DNA Detail of Restriction Site Fragments linked permanently by DNA ligase No break in DNA molecule The fragments are able to join together under the influence of DNA ligase.

Escherichia coli bacterial cell Using Plasmids Escherichia coli bacterial cell Human cell Preparation of the Clone Human gene Sticky end Plasmid Chromosome DNA in chromosome A gene of interest (DNA fragment) is isolated from human tissue cells Restriction enzyme recognition sequence Tetracycline-resistance gene Ampicillin-resistance gene Plasmid vector Plasmid vectors, found in bacteria, are prepared for cloning following steps 1-6: Human DNA and plasmid are treated with the same restriction enzyme to produce identical sticky ends Sticky ends Gene disrupted An appropriate plasmid vector is isolated from a bacterial cell Restriction enzyme cuts the plasmid DNA at its single recognition sequence, disrupting the tetracycline resistance gene Recombinant DNA molecule Human gene Mix the DNAs together and add the enzyme DNA ligase to bond the sticky ends Recombinant plasmid is introduced into a bacterial cell by simply adding the DNA to a bacterial culture where some bacteria take up the plasmid from solution

Activity Answer the questions on p237/8 in the Biozone books.

Gene Cloning – Making Copies Gene cloning = Process of making large quantities of a desired piece of DNA after isolation. Biologists obtain genes for cloning from two main sources: DNA isolated directly from an organism. Complementary DNA made in the laboratory from messenger RNA (mRNA) templates. Allows for an unlimited number of copies of a gene to be produced for analysis or for production of a protein product. Methods have been developed to insert a DNA fragment of interest (e.g. a segment of human DNA) into the DNA of a vector, resulting in a recombinant DNA molecule or molecular clone. Large quantities of the desired gene can be obtained if the recombinant DNA is allowed to replicate in an appropriate host.

Vectors for Gene Cloning Vector = self-replicating DNA molecule (e.g. plasmid or viral DNA) used to transmit a gene from one organism into another. All vectors must have the following properties: Be able to replicate inside their host organism. Have one or more sites at which a restriction enzyme can cut. Have some kind of genetic marker that allows them to be easily identified. Organisms such as bacteria, viruses and yeasts have DNA which behaves in this way.

Cloning the Gene The gene cloning process (making multiple copies of the human gene) occurs when the bacterium with the recombinant plasmid is allowed to reproduce. Colonies of bacteria that carry the recombinant plasmid can be identified by differential response to antibiotics Bacterium Human gene Recombinant plasmid All colonies look identical but only some have the plasmid with the human gene. Agar containing ampicillin allows only bacterial colonies with the appropriate plasmid to grow. Filter paper is pressed against the agar plate thereby transferring colonies of bacteria to the paper. The filter paper is pressed against agar that contains tetracycline. Those colonies that grow do not have the human gene disrupting the tetracycline resistance gene. Colonies with the human gene can be located according to their position on the original dish A. Bacteria containing the recombinant plasmid are spread onto an agar plate containing ampicillin. Dish A Filter paper Dish B