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Lesson 4 - Analysis of Lab Results to Verify Network Interactions

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Lesson Title: Lesson 4- Analysis of Laboratory Results to Verify Network Interactions 

 

Objectives: 
What students learn…
  • Scientists use computer programs and simulations to analyze networks because networks are often very complex.
  • One change in the environment could affect an entire cellular network due to the interdependent nature of networks.
  • A centrifuge separates a culture into a supernatant and pellet, which allows the phenotype of cells to be more easily viewed.
  • Qualitative measurement is a way of describing data with words, whereas quantitative measurement is a way of describing data with numbers.
  • Mutant strains, with known genotypes, are a useful tool when studying organisms.
 What students do…
  • Students use wild type and mutant strains of Halobacterium salinarum to investigate quantitatively and qualitatively.
  • Students use a centrifuge to spin down cells as a way to determine the phenotype of cells.
  • Students compare, contrast and critique divergent results from their investigations and discuss/identify possible errors/sources of variation.  
  • Students use a simulation to verify and/or correct their network understanding.
  • Students plan the steps needed for scientists to determine and verify a question using systems methods.
  • Students analyze the role the environment has on gene expression. In this analysis, recognize the components, structure, and organization of systems and the interconnections within and among them.

Instructional Activities: (Approximately three 50-min periods)

Before beginning lesson:         

Teachers  spin mutant broth samples into pellets at 6000 rpm for 15 minutes, pour off supernatant.
Pellet should be easily seen when given to students. Tip: cells will begin to dislodge and go into solution if not spun for 15 minutes (adjust time if spinning at higher rpm). There should be enough wild type and mutant samples to make a set for each team (8 teams per kit.)
This set of activities may play out differently depending on your facilities and equipment. We put the Mutants PowerPoint, Data Collection from the Student Experiment, and Simulation all in one part. They can be done in nearly any order and can happen somewhat simultaneously (esp. gathering O.D. data). Our description below is the way we think would be optimum. Please adjust to meet your needs.

1. Mutants PowerPoint(Lesson 4.ppt)       

a. Entry task: Have students entering room guess how the purple broth (screen slide 2) came to be. “How could we make this in our lab?” Discuss – accept all reasonable guesses (light, oxygen, enzymes). They don’t need to get to the perfect answers at this point (but at least close). [note for teacher: when bat is over expressed it causes halo to makes lots of bR, even without light.]
 
b. Slides 3 and 4. Explain to students what mutants are (forcibly expressed or removed genes) [note to teacher: this is a fairly easy/common procedure in labs now] and ask what Halobacterium mutants (bat +, bat -, bop-) would look like. They can use their networks (and the ppt. slide) to predict the color of these mutants. (have students record phenotypic predictions for each mutant in their notebook.)
 

c. Show the class the mutant pellets and this short (1/2 page) handout for

Have students record the color of the mutants and compare them to their predictions. Discuss any significant differences between predictions and the actual colors of the mutants. 

2. Collect Data from Student Experiments (This started in lesson 2)

Have students record the O.D. of their samples and spin down a pellet and record the color.

Color cubes.doc

 (Depending on number of spectrophotometers and groups this may take a while. To keep the samples comparable the entire classes’ experiments should all be removed from their heat source at nearly the same time. They can be left on the bench for up to 24 hours without much noticeable difference. If it will be any longer before they can take their O.D. the bacteria need to be refrigerated to keep them from continuing to replicate.) 

3. Simulation:  PM Network simulation ws &    PM simulation ws answers

Have students manipulate the simulation to verify their thinking about this network and how their data and the class data and the mutant data fit. (This is a good activity while waiting for O.D. data collection.)  *Teacher note: simulation requires JAVA, try a different browser if it does not load properly.

4. Compile Class Data

Help students gather the class data onto an excel spreadsheet. (There are multiple sheets for each template.)

Summary Data Template.xls

This is a good place to discuss how we took quantitative data and qualitative data (using the color cubes) and what the difference is and how to compare qualitative data to other qualitative data.

5. Analyze Class Data

Have students decide what the best way is to present the data they have gathered as a class. 

Light Dark OD Template.xls

Talk students through the data analysis process. It will be much easier if you decide to have the class work on only the light/dark data as a group. If your class worked on many different experiments, it might be hard to analyze it all together.
          a. analysis is not deciding what the data means, it is deciding what format will work best to show others the data.
          b. the class takes the raw data and turns it into summary data. This data is “prettier” and easy for others to look at and understand.
          c. sometimes it includes averages or trends or graphs

6. Student Review Sheet Lab Review and Analysis & Lab Review and Analysis-Answers

          a. the first section scaffolds the students through understanding their experimental data.
          b. the second section adds the information on mutants.
          c. the third section is a set of questions with a graph on light/dark and oxygen/no oxygen data.

7. Teacher prompts for discussions of information gathered from the network, the student experiments, and the mutants:

a. Compare the mutants (known) with student results (unknowns). Did the unknowns turn out as expected?
b. Phenotype = gene expression. Everyone in class has Halobacterium with the same genotype (clones), how can they have different phenotypes? What caused this? What causes this in nature?

c. Review the pictures from the introductory powerpoint: 

Environmental Influence on Gene Networks Introduction

Use the pictures that ask “Why?” (in the beginning only). Do the students have a better suggestion now to answer the “Whys”?
 
d. Now that we have a working network and some examples of mutants that validate our network, what is the next step? What do scientists do in this situation? (They present to their peers before publication) Then what do they do? (Scientists will look at the next question that comes up from their findings [ex. what network does Halobacterium use for energy when bacteriorhodopsin (bR) is not being produced or when there is a lot of Oxygen.])

8. Presentation

Student groups prepare a quick presentation of their findings and how they relate to the network they created (PowerPoint, brochure, document camera, oral presentation) include their data, class data, and the mutants. Each teacher can decide what format and how much time to spend on this part. Included is a sample of directions for a Powerpoint presentation. Halo Response to Environment PPT Format.doc & Halo Presentation Scoring Guide.doc

Assessment:  How will I know they know...

Post assessment (Post Assessment.doc & Post Assessment w-Answers.doc
Presentation of data

Please help us to make improvements to this module.  We would appreciate a few minutes of your time to take a short survey.   TEACHER  MODULE EVALUATION SURVEY and/or STUDENT MODULE EVALUATION SURVEY

Resources

Lesson 4.ppt      

Color cubes.doc

PM Network simulation ws

PM simulation ws answers

Summary Data Template.xls

Light Dark OD Template.xls

Lab Review and Analysis Worsheet

Lab Review and Analysis Answers

Environmental Influence on Gene Networks Introduction.ppt

E.I.G.N. Intro PPT Script

Halo Response to Environment PPT Format.doc

Halo Presentation Scoring Guide.doc

Post Assessment.doc

Post Assessment w-Answers.doc

Please help us to make improvements to this module.  We would appreciate a few minutes of your time to take a short survey.   TEACHER  MODULE EVALUATION SURVEY and/or STUDENT MODULE EVALUATION SURVEY

Accommodations: Students may have trouble deciphering the class data.  Have students seminar in smaller groups before having a whole-class discussion. 

Extension Activities:  Students engage in a virtual lab experience involving transgenic flies.  Focus is on experimental design and what scientist learn from mutation.  Go to http://www.hhmi.org/biointeractive/vlabs/transgenic_fly/index.html

 Short animations illustrating how gene expression results in phenotype changes.  http://www.hhmi.org/biointeractive/evolution/animations.html       

Students use bioinformatics (CLUSTALW and BOXSHADE) to explore why humans do not produce vitamin C (as most mammals do).  This three-part lesson series is called "Psuedogene Suite" and can be found here (the bioinformatics portion is in part 3): http://www.indiana.edu/~ensiweb/bioinfo.html