Students should be able to:
This set of laboratory exercises will acquaint you with molecular genetics and biotechnology. You will be modeling and manipulating DNA so you may initially understand its structure and chemical / electrical properties. You will also alter genetically alter a bacterium by inserting a plasmid (a circular strand of DNA containing specific genetic information) into selected bacteria.
You will be using various types of equipment, models and materials in order to carry out the lab activities for this exercise. The exercise will require three laboratory periods. You are to turn in the Pre-Lab Questions and the Contributions Sheet prior to lab. Make sure you have done all of the Pre-Lab Activities and have downloaded and printed out any materials, and have received from your instructor the handouts required for lab.
DNA / RNA
DNA (deoxyribonucleic acid) is one component of the "genetic material". It is composed of four different nucleotide units. A nucleotide is composed of a nitrogen base, (either adenine, thymine, guanine and cytosine), a deoxyribose sugar (C5 or pentose), and a phosphate group. The four nucleotides are named for their nitrogen base, hence they are an adenine nucleotide (A), a thymine nucleotide (T), a guanine nucleotide (G) and a cytosine nucleotide (C). These nucleotides monomers are arranged in particular sequences to form the larger double helix of the DNA polymer. There are several types of DNA found in living organisms.
RNA (ribonucleic acid) is also a polymer built of nucleotides. It is composed of four different nucleotides. Each of the RNA nucleotides is formed from the chemical combination of a nitrogen base, just as in DNA, except in RNA, the nitrogen base thymine is replaced with the nitrogen base uracil. The sugar is ribose instead of deoxyribose. The phosphate group remains the same as in DNA. The polymer is also different because it is a single helix and is very much shorter than DNA.
Transcription / Translation
The sequence of nucleotides in the DNA molecule can code for the sequence of amino acids in a protein. You will use modeling to help in your understanding of the central dogma of molecular genetics, that of passing the "code" from DNA to RNA to the "building" of a protein. The DNA "code" is transcribed into complementary mRNA (messenger RNA), which is translated into specific amino acids using tRNA (transfer RNA). This process of protein synthesis occurs in the nucleus of the cell (transcription) and then moves to the cytoplasm (translation).
Restriction enzymes will be used to fragment a sample of DNA (lambda DNA). Restriction enzymes are found naturally in bacteria and are useful in preventing a bacterium from being colonized by viruses (bacteriophages, DNA surrounded by a protein sheath). They "attack" the bacteriophage by cutting it into fragments thus the virus is no longer capable of colonizing the bacterium. The restriction enzyme works by cutting the DNA at a specific nucleotide sequence. There are many different restriction enzymes, each cut at a different sequence. One of the restriction enzymes that you will use is EcoRI. EcoRI stands for:
EcoRI "finds" and cuts at the DNA sequence:
The above represents a strand of complementary DNA where the letters represent the nucleotides.
EcoRI cuts between the G and the A nucleotide on both strands of the DNA. These cuts produce what is termed a "sticky end". Other restriction enzymes work in a similar manner.
Once DNA is cut it produces DNA fragments of varying length. These fragments can be separated from one another by using a technique called gel electrophoresis.
Gel electrophoresis is a technique that is widely applied in biotechnology. It is used to separate organic molecules, or fragments of molecules, which have an electrical charge on them (usually negative). These organic molecules can be proteins, DNA, RNA, etc. The process is carried out in a chamber that contains a buffer solution (usually TBE). The buffer solution conducts an electrical current, which produces an electrical field in the chamber. The chamber also contains a gel tray which contains a slab of agarose gel that has "wells" for solutions that contain the organic molecules that are to be separated from each other. When the chamber is electrified the negatively charged molecules migrate through the gel and are separated out in the gel. The rate of migration in the gel depends on the molecule's affinity for the current, it size and its shape. The migration of the organic molecules is toward the positive electrode. A picture of the gel electrophoresis equipment can be obtained during Pre-Lab Activity #3 or by clicking here.
Transformation is the process by which a plasmid is incorporated into a bacterium. This process occurs naturally in bacteria. Once biologists understood how the process worked and were able to duplicate it under controlled conditions in the laboratory a whole new area of biotechnology was established. Plasmids are circular extrachromosomal pieces of DNA that carry genetic information separate from the chromosomal DNA of the bacterium. Some plasmids can replicate autonomously and others only when the bacterial chromosome replicates. The plasmids that you will be working with are called R plasmids which carry genes for antibiotic resistance. The specific plasmid for this lab is one that contains a gene (Ampr) that confers resistance to the antibiotic, ampicillin. You will be using the bacterium Escherichia coli (E. coli) for this exercise and will, through a series of processes, transform nonresistant bacteria into resistant ones by making the nonresistant bacteria "competent" so they will absorb the plasmid, pAMP.
Pre-Lab Activity #1 - DNA Extraction
Pre-Lab Activity #2 - Colony Transformation Information
Pre-Lab Activity #3 - Restriction Enzyme and Gel Electrophoresis Information
Pre-Lab Activity #4 - Research
1. Using the resources available in the LRC (Access Science) find and printout the biographies of:
2. Watson and Crick's Original Paper - Nature, 1953
The Laboratory Activities and Data Collection
Lab Activity #1 - DNA Modeling
Lab Activity #2 - DNA Extraction
Lab Activity #3 - Modeling Transcription / Translation
Lab Activity #4 - Colony Transformation
Lab Activity #5 - Restriction Enzyme Fragmentation of DNA and Gel Electrophoresis of DNA Fragments
Post-lab Activity and Data Analysis
Results and Analysis:
Below are listed the activities you performed in lab and the results and analysis materials that are to be given to the instructor.