pGLO An Introduction to The World of Biotechnology

Sections What is Biotechnology? The GFP and the deep Blue Sea Transformation: Manipulating Bacteria Plasmids Putting the Process to work: The pGLO lab activity

What is Biotechnology? Biotechnology encompasses a field of ‘applied’ biology involving the use of living organisms in engineering, technology, and medicine for the betterment of society.

Biotechnology: Making the world a Better Place In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes the disease.

The Gene GFP Our Biotech journey starts with the biofluorescent jellyfish Aequorea victoria. Its unique Green Fluorescent Protein (GFP) causes the jellyfish to glow in the dark. Before we move on, it is essential that you review and understand the central dogma of molecular biology….

Bacterial Plasmids In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to the survival of the organism. Bacteria easily transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments.

Transformation Genetic transformation involves the insertion of a gene into an organism, resulting in genotypic (and phenotypic) change in the modified organism. Why are bacteria ideal vectors for genetic transformation?

“WAIT!.... What makes this possible?”

Transformation Specifics: Gene Splicing and palindromes in DNA Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they operate within the molecule itself, they are often called restriction endonucleases. Specialized restriction enzymes cleave DNA molecules at precisely-located sites so that a small set of identical fragments are produced. The rarer the site it recognizes, the smaller the number of pieces produced.

Manufacturing Recombinant DNA Restriction enzymes seek out, or ‘recognize’ unique palindrome sequences within DNA (regions that are the same forward and backward) and precisely cut sequences with ‘sticky ends.’ New DNA segments with the desired gene(s) can be then ‘pasted’ into these ‘sticky’ regions, creating recombinant DNA, and ultimately a genetically modified organism. A man, a plan, a canal: Panama.

The pGLO Plasmid Bio-Rad’s engineered pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin. This special pGLO plasmid also incorporates a unique gene regulation system, used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be ‘switched on’ in transformed cells by adding the sugar arabinose to the cells’ nutrient medium.

Your Task… In this lab, YOU will utilize biotechnology tools, techniques, and restriction enzymes to insert Biorad’s special genetically modified pGLO plasmid into e coli bacteria. If done properly, YOU will create a genetically modified organism expressing recombinant DNA!

The pGLO Lab Activity The website below contains the student manual for the lab activity. It is essential that you review proper techniques with regard to the use of micropipettes before you start the lab. Additionally, you MUST carefully review the procedures (as well as the reasons why they’re implemented) BEFORE you begin the pGLO lab. GOOD LUCK! http://www.biologyforlife.com/Units/Year%201/Genetic%20Engineering/lab%20pdf.pdf

pGLO DO’s and DON’Ts DON’T DO Skip steps Forget to use proper technique and/or replace tips and loops during transfer Screw up DO Work through the lab step by step Use proper sterile techniques Ask for help/clarification when you need it