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.
Mutants PowerPoint (Lesson 4 PowerPoint | Google Slides – coming soon)
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 Pellet background information. 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.
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 (Google Doc | Word 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 halobacteria need to be refrigerated to keep them from continuing to replicate.)
Simulation: Purple Membrane (bacterioRhodopsin) Network Simulation Student Worksheet & Instructions (Google Doc | Word Doc) and PM Simulation WS Answers (Google Doc | Word Doc)
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.) This activity also directly addresses the Next Generation Science Standards for Systems and Systems Models.
If the online simulation is not working, you can also download the needed free software (NetLogo) and file (purple_membrane_5.3.1.nlogo) to run this simulation.
- Instructions (if the online simulation is not working:
- Download, run, and/or save NetLogo 5.3.1 onto your PC, Mac, or Linux machine.
- Download this zipped file: purple_membrane_5.3.1.
- Extract the file from the zipped folder by either dragging it into a new folder or saving it into a new folder. Do not change the file name from “purple_membrane_5.3.1.nlogo”.
- Launch NetLogo 5.3.1 from your machine.
- Navigate to the “File” tab and then select the “Open” subsection.
- Browse and select the “purple_membrane_5.3.1.nlogo” file you saved.
- Click the start button to begin using the model. Or follow the instructions included in the worksheet to use the network model.
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.
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
Student Review Sheet Lab Review and Analysis (Google Doc | Word Doc) & Lab Review and Analysis-Answers (Google Doc | Word Doc)
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.
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:
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.])
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 (Google Doc | Word Doc) & Halo Presentation Scoring Guide.doc (Google Doc | Word Doc)
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