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Part I. Manipulation of DNA
Part I
Manipulation of DNA The goal of these laboratory exercises is to fuse a jellyfish gene with a bacterial gene and to express a single protein from this hybrid DNA sequence. Why would you want to do this? Molecular shuffling of genetic sequences, or gene cloning, is a powerful tool for understanding biological processes and for biotechnological applications. Using basic tools developed in Escherichia coli, we can ask questions about other, more complicated organisms. Scientists have exploited E. coli both as a workhorse for producing DNA and as a source of well-characterized sequences to direct the transcription and translation of foreign DNA into protein. With genetic sequence information being produced at a breathtaking rate, the limiting factor is not in sequencing DNA, but in our understanding of the function of the products of these sequences. In terms of practical biotechnology applications, it can be a huge advantage to clone the gene encoding a difficult-to-purify protein into E. coli so that the purification process can be accomplished less expensively and to a greater degree of purity (and oftentimes more ethically, especially if a human gene is involved!). The first recombinant protein to be produced and marketed was human insulin in the early 1980s, which has been invaluable to countless diabetics. The basic tools you will learn in this class will enable you to clone, express, and purify recombinant proteins. They will enable you to begin to probe the function of any protein for which a gene has been identified, and will give you the conceptual background needed for tackling more advanced techniques. Other hosts are now commonly used for cloning DNA and expressing recombinant proteins, such as members of the bacterial genus Bacillus, as well as eukaryotic hosts including numerous species of yeast and other fungi, plants, insect cell culture, mammalian cell culture, and even whole, live mammals (“pharming”). Many of the recombinant DNA methods used in this course are applicable to cloning in other hosts. The gene we will be cloning and expressing is the enhanced green fluorescent protein gene, egfp. EGFP is a brightness-enhanced variant of the green fluorescent protein from the jellyfish Aequorea victoria1. The gene encoding the green fluorescent protein (and its variants, including the egfp gene) is widely used as a “reporter gene” or “marker.” A reporter gene is a gene that is used to track protein expression. It must have phenotypic expression that is easy to monitor and can be used to study promoter activity or protein localization in different environmental conditions, different tissues, or different developmental stages. Recombinant DNA constructs are made in which the reporter gene is fused to a promoter region of interest and the construct is transformed or transfected into a host cell or organism. EGFP can also be used to mark (or tag) other proteins by creating recombinant DNA constructs that express fusion proteins that fluoresce and can be tracked in living cells or organisms. In this project, we are not using egfp as a reporter, but rather as a convenient gene to clone and assay for, as we learn the basic techniques of recombinant DNA manipulation and protein expression.
2
PART | I Manipulation of DNA
REFERENCE 1. Yang T, Cheng L, Kain SR. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 1996;24(22):4592 4.
Manipulation of DNA The goal of these laboratory exercises is to fuse a jellyfish gene with a bacterial gene and to express a single protein from this hybrid DNA sequence. Why would you want to do this? Molecular shuffling of genetic sequences, or gene cloning, is a powerful tool for understanding biological processes and for biotechnological applications. Using basic tools developed in Escherichia coli, we can ask questions about other, more complicated organisms. Scientists have exploited E. coli both as a workhorse for producing DNA and as a source of well-characterized sequences to direct the transcription and translation of foreign DNA into protein. With genetic sequence information being produced at a breathtaking rate, the limiting factor is not in sequencing DNA, but in our understanding of the function of the products of these sequences. In terms of practical biotechnology applications, it can be a huge advantage to clone the gene encoding a difficult-to-purify protein into E. coli so that the purification process can be accomplished less expensively and to a greater degree of purity (and oftentimes more ethically, especially if a human gene is involved!). The first recombinant protein to be produced and marketed was human insulin in the early 1980s, which has been invaluable to countless diabetics. The basic tools you will learn in this class will enable you to clone, express, and purify recombinant proteins. They will enable you to begin to probe the function of any protein for which a gene has been identified, and will give you the conceptual background needed for tackling more advanced techniques. Other hosts are now commonly used for cloning DNA and expressing recombinant proteins, such as members of the bacterial genus Bacillus, as well as eukaryotic hosts including numerous species of yeast and other fungi, plants, insect cell culture, mammalian cell culture, and even whole, live mammals (“pharming”). Many of the recombinant DNA methods used in this course are applicable to cloning in other hosts. The gene we will be cloning and expressing is the enhanced green fluorescent protein gene, egfp. EGFP is a brightness-enhanced variant of the green fluorescent protein from the jellyfish Aequorea victoria1. The gene encoding the green fluorescent protein (and its variants, including the egfp gene) is widely used as a “reporter gene” or “marker.” A reporter gene is a gene that is used to track protein expression. It must have phenotypic expression that is easy to monitor and can be used to study promoter activity or protein localization in different environmental conditions, different tissues, or different developmental stages. Recombinant DNA constructs are made in which the reporter gene is fused to a promoter region of interest and the construct is transformed or transfected into a host cell or organism. EGFP can also be used to mark (or tag) other proteins by creating recombinant DNA constructs that express fusion proteins that fluoresce and can be tracked in living cells or organisms. In this project, we are not using egfp as a reporter, but rather as a convenient gene to clone and assay for, as we learn the basic techniques of recombinant DNA manipulation and protein expression.
2
PART | I Manipulation of DNA
REFERENCE 1. Yang T, Cheng L, Kain SR. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 1996;24(22):4592 4.