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Chapter 10: Biotechnology. Part 1. Frankenfood ??. Scientists are now able to insert genes from one species into another.
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Chapter 10: Biotechnology Part 1
Frankenfood?? • Scientists are now able to insert genes from one species into another. • In one example, scientists have inserted genes from a daffodil and a soil bacterium into a rice plant, enabling the rice plant to produce beta-carotene in its edible parts, which can then be metabolized by the human body to generate Vitamin A. http://en.wikipedia.org/wiki/Golden_rice
Frankenfood?? • This is especially important globally since Vitamin A deficiency causes 500,000 cases of irreversible blindness and up to 2 million deaths each year, especially in pregnant women and children. • Across the globe, around 19 million pregnant women and 190 million children suffer from Vitamin A deficiency because people in developing countries do not have foods rich in Vitamin A, such as vegetable, fruits, and animal products, in their diets. http://www.goldenrice.org
Frankenfood?? • Golden rice, like many other transgenic organisms, are also called genetically modified organisms or GMO’s. • This simply means that it contains genes from a different species. • Though these organisms are not produced on farms, their production involves an extension of breeding practices that have been used for thousands of years to produce new plants and new species of animals from wild species.
Frankenfood?? • No one wants millions of women and children to die from Vitamin A deficiency, yet many people are opposed to GMO’s. • They feel that out ability to tinker with genes far surpasses our ability to understand the impact that our tinkering may have on ecosystems. • Should we be more cautious? • How much of a risk should society take to help dying women and children?
Frankenfood? • As we will see, geneticists already hold the molecular keys to unlocking the kingdom of inheritance and their endeavours are already having an impact on our biosphere.
Cutting and Pasting • After the discovery of the structure of DNA in the 1950’s, there was excitement followed by frustration as scientists began to discover how impossible it seemed to locate a specific base or gene among thousands or millions in a DNA molecule. • The discovery of restriction enzymes changed all of that.
Cutting and Pasting • Restriction enzymes are enzymes used by bacteria to protect themselves from viruses that inject their DNA into bacterial cells. • These enzymes cut the viral DNA into pieces, rendering it unable to infect the bacterial cells. • Amazingly, these enzymes can cut DNA from any source.
Cutting and Pasting • Each restriction enzyme has its own specific base sequence that it can recognize, called pallindromic sequences. • They are so called this because they are read the same forward on one strand as they are backward on the other strand. • It then cuts the DNA at this sequence, wherever it occurs in the sample of DNA. • For example, EcoR1 (a restriction enzyme isolated from E. coli), recognizes and cuts DNA only at the sequence GAATTC.
Cutting and Pasting • Some restriction enzymes produce blunt ends while others produce sticky ends. • Enzymes that produce sticky ends are the most desirable. • If the same restriction enzyme is used to cut both the target DNA (ie. rice plant DNA) and the source DNA (ie. daffodil and soil bacterium DNA), the sticky ends from all the DNA (both source and target) will have sticky ends that are complementary to one another and so they will stick to each other when mixed together in the same tube with DNA ligase (an enzyme that reforms the hydrogen bonds between complementary nitrogen bases). • The DNA produced by this process is called recombinant DNA. • In this way, scientists were able to “stick” DNA from the daffodil and the soil bacterium into the rice DNA.
Cutting and Pasting Rice plant DNA Daffodil DNA
Cutting and Pasting • Making recombinant DNA is the first step in DNA cloning, a lab process that uses living cells to produce many copies of a specific DNA fragment. • Researchers often insert DNA fragments that they have produced using restriction enzymes into plasmids. • Plasmids are small circular molecules of DNA (independent of the bacterial chromosome) that contain genes that bacteria may use in times of stress to aid in their survival.
Cutting and Pasting • When bacterial cells reproduce they also copy and pass on their plasmids to descendant cells. • Any fragment of foreign DNA that has been inserted into the plasmid will also get copied as part of the plasmid and passed on to descendant cells. • Thus, plasmids can be used as cloning vectors (molecules that carry foreign DNA into target cells).
Cutting and Pasting • So, how do researchers get the recombinant plasmid that they have created into the bacterial cells? • They subject the cells to some environmental stress, such as extremely hot temperatures, for a short period of time. • When subjected to this stress, bacteria will scan the environment for any plasmids that might contain genes that would help them to survive the stress. • Exposing bacterial cells to hot temperatures (called heat shock) can induce at least some bacterial cells to take up the plasmid containing recombinant DNA.
Cutting and Pasting • A target cell that takes up a cloning vector such as a plasmid can be grown in the laboratory to produce a huge population of genetically identical cells called clones. • A target cell that has taken up the plasmid containing the foreign DNA has been transformed. • The process of a bacterium taking up a plasmid is called transformation. • Not all target cells will take up the plasmids containing the foreign DNA (which we will call gene of interest from here on).
Cutting and Pasting • How do researchers select only for those cells that were transformed? • They also insert on the plasmid containing the gene of interest, another gene that will code for a trait that can be selected for, such as an antibiotic resistance gene or a gene that will cause the bacterium to change color when grown on certain culture media. • When the bacteria are grown on the special culture media, only the ones that have been transformed will survive or change color. • In this way, scientists can select for only the bacterial cells that have been transformed.
Cutting and Pasting • Once scientists have selected for the cells that were transformed, they can select only these cells to continue to culture, allowing the cells to reproduce by binary fission to produce thousands or more cells, all containing the recombinant plasmid with the gene of interest. • In order to ensure that the transformed cells keep their plasmid, scientists must continue to grow the cells on the special media that was used to select for the transformed cells. • If the cells were grown on regular media, they would dispose of their plasmid. • This is because bacterial cells only keep their plasmids as long as they need them. • When the plasmid is no longer needed for survival, the bacterium releases it into the environment.
Cutting and Pasting • Why would we want a bacterium to take up a recombinant plasmid? • We can cause the bacterium to produce a gene product that we want, then isolate the product from the bacterial cells.
Cutting and Pasting • For example, bacterial cells that have been transformed with a recombinant plasmid containing the human insulin gene now produce human insulin. • This insulin is sold under the name Humulinor Humulog. • Before this, diabetics had to take cow or pig insulin and many had allergies to this insulin because it is not exactly like human insulin.
cDNA Cloning • Many times researchers who study eukaryotic genes and their expression work with mRNA. • mRNA, however, cannot be cloned directly because restriction enzymes and DNA ligase can only cut and paste double stranded DNA. • However, mRNA can be used as a template to make double-stranded DNA in a test tube.
cDNA Cloning • An enzyme called reverse transcriptase can assemble a strand of complementary DNA (cDNA) on an mRNA template • When DNA polymerase is added to the mixture, it strips the mRNA from the hybrid molecule and makes a second strand of DNA complementary to the cDNA strand. • The end product is a double-stranded DNA copy of the mRNA molecule. • This double-stranded DNA molecule can then be cut with restriction enzymes and pasted into a cloning vector (such as a plasmid) using DNA ligase.
Questions • Why is it necessary to “heat shock” bacteria to cause them to take up a plasmid? • Why do we want the bacterium to take up a plasmid? • Why would we want to clone cDNA?
