labs

=Lab activities for July 9-20= Some of the background information for our lab activities is posted here. Use the Discussion to post questions, suggestions, etc.

Monday July 9

 * DNA isolation** - Isolating DNA from cells or tissue is the first step in many biotechnology processes. There are many protocols available to isolate DNA from a variety of living organisms, using common household reagents (detergents, salt, alcohol). You will work in groups to isolate DNA from a particular type of tissue, in an inquiry-based format where some variable in the process will be tested. See "[|How to Extract DNA from Anything Living]" from the University of Utah site for an overview of the general process required to get DNA away from everything else in the cells. Here are a few protocols developed for specific DNA sources:
 * [|Wheat germ]
 * [|Onion] (pdf) (also used with other vegetables and fruits)
 * [|Liver] (other organ tissues such as thymus (sweetbreads) and testes work well if you can get them; muscle does not work as well)
 * [|Yeast]
 * Here is a protocol for starting with purified, purchased DNA, to demonstrate spooling and the expected appearance of DNA:[[file:DNA spooling_instructor.pdf]]

Each group will pick a starting tissue and design a protocol. They will also choose a variable to test (a step in the protocol to vary, volumes or concentrations to alter). After isolating the DNA, you will evaluate which protocols worked best. (How will we define "worked best"?). We will have materials available for these protocols and some extra miscellaneous supplies. If you have done this before and have a favorite protocol, bring it along (or post it here). You are also welcome to bring any materials you'd like to try to isolate DNA from.

Tuesday July 10

 * Bacterial transformation** - We will demonstrate genetic engineering of bacteria by transforming //E. coli// with a plasmid containing the gene encoding green fluorescent protein (GFP) from jellyfish. Again, there are many kits and protocols available for bacterial transformation. We will be using the Bio-Rad kit; the kit protocol is here: [[file:pGlo-kit.pdf]] . We will prepare the reagents in the morning, and run the protocol in the afternoon (the agar plates need to sit a while to solidify). One interesting aspect to this particular kit is that it adds the concept of gene regulation. The pGLO plasmid includes a part of the //E. coli// chromosome containing genes encoding enzymes involved in metabolism of the sugar arabinose. These genes are only expressed in the presence of arabinose, which activates transcription from that promoter. In the plasmid, some of the arabinose metabolism genes are replaced by the GFP gene, which is then under control of the same promoter. So even if the bacteria contain the GFP gene, they will not glow unless the medium contains arabinose.


 * Identification and isolation of amylase-producing microorganisms** - Another important aspect of biotechnology is the identification of naturally-occurring organisms (often microorganisms) that have desirable characteristics. We will isolate soil bacteria that produce amylase, an industrially important starch-degrading enzyme. A protocol similar to the one we will be using is found at the [|Access Excellence] site. This one starts with homemade compost, but we will use soil samples from around campus (or you can bring in your own, if you like)

Wednesday July 11

 * Yeast genetics.** The budding yeast, //Saccharomyces cerevisiae//, is arguably the most important microorganism in biotechnology. Yeast have been used for biotechnological purposes throughout recorded history. They were first used (and continue to be used) for food biotechnology, producing fermented food and drink or as leavening agents for baking. Yeast fermentation has seen greater commercial use recently with the growing emphasis on ethanol based fuels. See [|GM yeast1]and [|GM yeast2] to learn how genetic engineering is being used to enhance ethanol production. Yeast are simple eukaryotic cells that are ideal for genetic analyses. Scientists have used this powerful model organism to learn much about how all eukaryotic cells function. For example, see [|yeast and cancer] to find out how yeast were used to identify genes that control whether or not our cells divide and become cancerous.

Yeast are well suited for teaching. The [|GENE] website from Kansas State University compiles information and resources for using yeast in the classroom. The site includes a wide array of laboratory exercises. In the morning lab we will begin a modified version of the [|Simple Cross Experiment]. Each group will be assigned an “unknown” yeast strain and asked to play detective. We will use simple genetic tests including nutritional selection and mating to help identify the unknown yeast. We will monitor the results of these experiments over the next few days (yeast take some time to grow). This lab is designed to illustrate some basic genetic principles as well as to give you hands on experience working with yeast.


 * Food biotechnology.** The afternoon lab will focus on food biotechnology. We will demonstrate fermentation by preparing yogurt. Yogurt is fermented milk. Active (unpasteurized) yogurt cultures contain the bacteria //Lactobacillus bulgaricus// and //Streptococcus thermophilus.// These bacteria possess enzymes that allow them to convert milk sugar (lactose) into lactic acid. Lactic acid is what gives yogurt its sour flavor and is also what causes the milk proteins to curdle (denature). We will inoculate milk with small amounts of active yogurt culture and monitor the fermentation (how?). Groups will explore how differences in culture conditions influence the fermentation process.

Here is fermentation unit developed by teachers attending a biotechnology workshop at UWRF; it includes several inquiry-based activities involving various types of fermentation.

Thursday July 12

 * Amplification of DNA by PCR and agarose gel electrophoresis.** The development of the polymerase chain reaction (PCR) for amplifying specific regions of DNA has revolutionized biology research and applications such as diagnostics. A history of PCR and background information can be found at the [|Access Excellence] site. To demonstrate this technology, we will isolate DNA from your cheek cells and amplify a sequence called an //Alu// insert. About 11% of the human genome is composed of these //Alu// inserts, which are transposable elements that can "jump" around the genome by copying themselves and inserting in new regions of the genome. The presence or absence of //Alu// inserts can be used to identify DNA in forensics cases, and they are also useful in determining evolutionary relationships. We will detect the presence of an Alu insert called PV92, located on chromosome 16. This Alu is not near any structural genes and is not associated with any phenotype, so it is appropriate to use in a classroom setting where students will test their own DNA. Background theory and an overview of the protocol are at the Cold Spring Harbor [|DNA Learning Center] site. The size of the DNA fragment amplified is determined by agrose gel electrophoresis.

Manual for Bio-Rad GMO detection kit
 * Detection of GMO foods.** Bio-Rad cells a kit for using PCR to detect the presence of genetically modified organisms (GMO) in food samples, and as it happens we have some leftover kit materials available.. This kit amplifies a promoter sequence commonly used when genes are transferred into plants, so it will detect BT-corn, Round-Up-ready soybeans, etc. If you would like to try this out (in additional to the human cheek cell PCR), bring in a sample of some food product that you think will (or will not) have GMO.

Friday July 13
Prepare and stain karyotype slides that you can take home with you, using a kit from [|CellServ]. Chromosomes are the central organizing structure for genetic information. The survival of an organism depends critically on the ability of its cells to organize and separate chromosomes during mitosis, so each new cell has the correct number. A recent Scientific American article describes a theory that is gaining support, regarding abnormal chromosome number as being the primary cause of a cell becoming cancerous, rather than mutations in specific genes. (As a side note, the author of this article, Peter Duesberg, is a well-known HIV denialist who has been ostracized by much of the mainstream biomedical community; Scientific American included an editorial justifying their publication of an article by this author. Read the article and the editorial here:, ) Genomic information is also organized by chromosome; genes are mapped and ordered by chromosome location. Being able to see these structures in a microscope may make some of the concepts more concrete.
 * Preparing chromosome spreads from cancer cells**

You can also generate a virtual karyotype from a chromosome spread at the [|University of Utah Genetics site].


 * Genetic Variation.** Our genomes are all very similar. Our DNA sequences are estimated to be over 99% identical. There are differences in our DNA and you saw an example of one yesterday. The relatively few differences in our DNA, called polymorphisms, are what make us unique. Polymorphisms are also what permit a wide variety of different DNA testing procedures. DNA tests can now be used to solve crimes, identify parents, diagnose diseases, customize treatments, and predict future ailments. Students are keenly aware of DNA testing, thanks in part to television shows like Crime Scene Investigators. We will share an approach that we have used to harness this interest in the classroom to help students understand how DNA testing actually works. We will analyze the results from some DNA based paternity tests that were performed by UWRF students. Check out [|who's your daddy] to view some of the candidate fathers. You might also want to check out the [|adventure diary] at this site to learn how Tasha, a former kindergarten teacher, now spends her time.


 * Lessons from the Human Genome.** The complete DNA sequencing of the human genome in April 2003 has been heralded by many as the single, greatest scientific feat of the decade. It was the "moon landing" of the 21st century. Like our first space exploits, it was not entirely clear what scientists hoped to discover from this DNA sequencing. We will discuss what scientists have learned from this achievement, some of the interesting new questions the sequence has raised, and ways that we can present this information in the classroom. The [|DOE genome website] contains a wide variety of educational resources related to the human genome.

The story of how the human genome was sequenced is fascinating. It highlights the complex interplay between public and private research that is increasingly common in biotechnology. Looking for some good books to read this summer? Check out [|The Genome War] and [|The Common Thread] for two very different accounts of this saga. We will use the story behind the sequencing to foster a discussion of some social and ethical issues associated with genomes and privatization.

Monday July 16
The morning lab will involve plant tissue culture using African violets. See [|PlantTC] for links to resources on plant tissue culture.

Tuesday July 17

 * RNA interference -** Today we will explore the exciting world of RNA interference and reverse genetics. RNA interference (RNAi) refers to the post-transcriptional silencing of a specific, targeted gene. RNAi has revolutionized modern biology by making it extremely easy to eliminate the function of a chosen gene. RNAi permits research scientists to ask questions about an individual genes function. RNAi may also be used in the future to cure heritable human diseases. RNAi was discovered accidently, by scientists studying a small worm. These scientists were awarded the [|Nobel Prize] in 2006 to recognize the broad impact of their discovery.

RNAi acts on mRNA, not DNA. RNAi prevents protein from being made from the targeted mRNA. Usually RNAi causes the mRNA to be degraded, but in some cases the mRNA is sequestered away from the translational machinery. Regardless the protein is not created. You can read more about this process at the [|Dolan Learning Center] (scroll down to where it reads “RNAi-nformation”).

//C. elegans// is a small nematode (worm) that lives in the soil. This worm is probably one of the most well understood animals on the planet. Scientists know the precise origin and fate of every worm cell during development. Check out [|worm link]to see movies of normal and mutant worm development. The worm genome is fully sequenced and perhaps more importantly, we now know the function(s) of most of those genes. RNAi has allowed scientists to conduct reverse genetics experiments on nearly every worm gene. Today we will conduct our own RNAi experiments.


 * Tissue engineering and stem cell cultures** - info to be added

Wednesday July 18

 * //In vitro//** **fertilization of //Xenopus laevis// eggs.** The African clawed frog, //Xenopus laevis//, has played many roles in biotechnology. You might be surprised to learn that frogs were once used to test for human pregnancy, or that the first cloned animal was in fact a frog. Frogs are extremely sensitive to changes in water quality and are being used as biomonitors for pollutants. New antibiotics have been discovered in frog skin. Frogs are a powerful model system for studying animal development. Female frogs contain lots of eggs and these eggs are quite large. Frog eggs are fertilized and the embryos develop outside the mother, allowing scientists and students to monitor this magical process with only minimal magnification.

In the morning lab we will explore the world of reproductive biotechnology by fertilizing frog eggs //in vitro// and watching the embryos develop. You can find a description of the //in vitro// fertilization procedure and other useful frog information at the [|Xlaevis] website. //In vitro// fertilization (IVF) is a large and growing biotechnology industry. IVF is now routinely used in agriculture to reproduce domestic livestock, and it is starting to be used to create other animals including [|endangered species]. Over a million human offspring have now been conceived through IVF. We have come a long way from Louise Brown, the first “test tube” baby.

It is often desirable to preserve gametes (typically sperm) for later use in IVF. The most effective way to store sperm or any eukaryotic cell for extended periods of time is to use extremely cold temperatures (cryopreservation). Special storage solutions are required for sperm to survive these cold temperatures. Unfortunately, different storage solutions are needed to preserve sperm from different species. There is currently no known method for storing frog sperm for later use in IVF. Each group will design their own frog sperm storage solution. We will provide reagents that are commonly found in cryopreservation solutions. Groups will then use microscopes to examine the effect their solution has on sperm motility.

Reproductive technologies such as cloning and stem cells raise many ethical issues. We’ll spend time in the morning sharing ways that we can effectively address these matters in the classroom.

You can order frogs for your own class as well other instructional material from [|Nasco].


 * Antibodies as tools**. Antibodies are proteins produced by mammalian white blood cells (specifically B cells) as part of an immune response against foreign invaders (viruses, bacteria, etc., generically known as antigens). The B cells secrete the antibodies, which bind to and neutralize the invader. A key feature of antibodies is their specificity - they will only bind to the antigens against which they were generated - and this makes them very useful tools in diagnostics and other applications. We will demonstrate three types of antibody-based test:
 * HIV test (ELISA) - The standard HIV test detects antibodies to HIV using an indirect ELISA. The the [|Arizona Biology Project] site for an animated tutorial of how the HIV ELISA works. We will be simulating that test (i.e. not using actual HIV proteins or real blood samples) using this protocol: [[file:HIV test instructor.pdf]]
 * Detection of a food allergen (immunoblot) - The presence of the wheat gluten protein, gliadin, which is responsible for many wheat allergies, can be detected in flour samples using a simple immunoblot. Flour extracts are spotted onto a membrane, and enzyme-labeled antibodies to wheat gliadin are added. Spots containing the allergen will produce color on the membrane. We may not have time to actually do this lab, but here are the instructions: [[file:wheat_immunoblot.doc]]
 * Pregnancy test (rapid test) - The pregnancy tests that you can buy at the drugstore use antibodies to human chorionic gonadotropin, produced by developing fetal cells, to detect pregancy. See and animation of how these tests work at our [|Immunology textbook web site.] (Click on Animations, and then click on hCG pregancy test under Chapter 6 in the left panel.)

Thursday July 19

 * Computer simulations of molecular biology techniques** - The [|Case It project] at UWRF, funded by the National Science Foundation, provides free software simulations for educators. The current version of the software simulates restriction enzyme digestion, gel electrophoresis (DNA and protein), ELISA, Western blot, Southern blot, dot blot, PCR. The program reads actual DNA or protein sequences, and generates results very similar to what is achieved in the lab - so it can be used in classes where the actual lab equipment is not available. We have packaged sequences for case studies in human genetics and infectious diseases that emphasize ethical and social issues. For the HIV case studies, we have video clips of people living with HIV. There are detailed tutorials for the software on the web site, where the software can be downloaded.


 * Bioinformatics** - We will also go through the bioinformatics activity handed out on Fri 7/13 (the packet with the dog DNA gel on the front), which uses the amylase gene as a model. This is from [|Recombinant DNA and Biotechnology: A Guide for Teachers], by Kreuzer and Massey, which has a number of activities that don't require a lot of equipment (e.g.paper versions of PCR). (We ended up not doing this activity, but rather just a quick introduction to the NCBI web site. Here is a summary of what we did (Thanks, Nathan!))