Lesson 3 – Data Analysis to Propose Network Function
Course: Biology, Genetics, Biotechnology, Environmental Science
Unit: Genetics and Heredity
- Cellular networks allow cells to respond dynamically to stimuli.
- A response to a stimulus occurs by changing the relative amount or function of individual nodes in a cellular network.
- A stimulus affecting one node can cause a change in many other nodes because they are all interconnected in the network.
- Some nodes have bigger system-wide affect than others.
- Understanding relative importance of nodes allows greater understanding of how to perturb and use the system.
- Multiple data sets from various experimental techniques are needed to show network relationships.
- Students analyze three different data sets to determine the bacteriorhodopsin network and to determine the importance of compiling multiple sets of data.
Instructional Activities: (Two 50-minute periods)
- Helpful animation showing enzyme function (you may need to copy and paste the link into a browser window to view the animation) - http://highered.mcgraw-hill.com/sites/0072495855/student_view0/chapter2/animation__how_enzymes_work.html
- Also, if students need a broad level overview of genes and/or the genome, this 5 minute animation is a terrific resource: The Animated Genome created by Sanan Media in partnership with the National Human Genome Research Institute. While this covers the human genome, one of the reasons we use Halobacterium as a model organism is because it processes its genome in a way that is similar to humans.
Slide 1 – Asks the question...How will you know what your lab results mean? Students will receive population and phenotype data, so they can support or refute their narrow hypothesis. What is important for this lesson is that they know this is not where an experiment ends.
° Prompt: Once you have supported/refuted your hypothesis – what is next?
If the students say – “Do another experiment” – ask: how do you know your first experiment makes sense (what if your data is totally invalid)? How do you know what ‘next experiment’ to do? If students say “Repeat it”, ask what happens if the data is the same….or different. Again, how do you know your first experiment makes sense (what if your data is totally invalid)?
° Get students to the idea that once an experiment is complete, scientists will then see how their data fits into the most current understanding of the organism. Some groups may even collaborate by comparing data and results.
Slide 2 – Asks, what did you measure? Answers should be: Color and Population Size
Asks, What are some things the data requires you to understand? (What is needed in order to make sense of the data?) This can go in many directions, but the teacher should remind students of what they know about color (caused by a pigment used in energy production) and cell division (requires energy).
Slide 3 – Asks, can difference in color be due to genes? Ask students for examples. *Be sure you discuss not just gene variation which can lead to differences in color (like siblings having different hair color), but also the status of those genes (their regulation) can change the phenotype (remind them of the tan line or hydrangea example from Lesson 1 Intro.
° Click – a diagram pops up to show how, in the presence of a certain enzyme, a colorless compound is converted into a red pigment molecule – giving a pink flower. Remind students of how the proteins in an organism relate to its physical appearance and how a lot of this action is an indirect relationship. This is especially true of enzymes that allow biological reactions to occur. If students don’t seem to understand, remind them that this DNA is in every other cell of the plant, so... Why are only parts of the petal turning pink and no other portions of the plant? Answer – in the red parts of the petal the gene making this enzyme is activated (turned on and actively transcribing), in other areas it is off. The “environment” of those cells flip on the enzyme making genes.
*POINT: homology data (scientist compare proteins of known pathways to see if the ones they are studying are similar) may help determine what is involved in gene expression resulting in a color change.
Next… What might help explain population growth?
Slide 4-6 - population (growth due to cell division) is connected to energy. Students should learn about current research on pathways of energy production (metabolism data) to help explain their data.
° During the jigsaw activity, students will look at an energy formation pathway (halo use bR to produce ATP using light energy in Halo which will allow them to get a better idea of whether their results “make sense”.
Slide 7 - shows the GSL and asks “what makes halo purple?” This is an opportunity or remind students that they are experimenting with both color and population growth so the jigsaw exercise will offer 3 different types of data that could help them answer confidently.
Slides 8 and 9 - shows a diagram of halo’s membrane with purple protein embedded. Click and a box shows around 1 of the purple proteins. Click again and it enlarges.
Slide 10 - asks what the purple protein is actually made of.
Slide 11 - shows it is called bacteriorhodopsin (bR).
Slide 12 - shows retinal and bop inside the cell. Click and they move together to form bR.
Slide 13 - asks what purpose of bR. Allow students to answer.
Slides 14 and 15 - answer (converts light…). Click and light is added. Click again and box appears around 1 of the purple proteins. Click again and it enlarges. * Note that light does not become bR, light increases the amount of BR.
Slides 16 and 17 - click and 2 ATP are made.
Slide 18 - diagram compares ATP production when light is present and absent.
Slide 19 - point out halo in light (left side) has many more purple proteins in membrane than halo in the dark (right side).
Slide 20 - by now students may ask (hopefully) about why there would be more bR in the membrane when light is present. If they haven’t arrived at this, the slide 20 asks it for them.
Slides 21 to 23 - illustrate the answer (increases gene expression..initiates transcription). Students will see this during the jigsaw exercise in more detail.
Slide 24 - the final slide states the guiding question for the lesson: “How do halobacterium cells change the amount of bR in response to light?” Explain to the students that they will be modeling what is done in science today (especially when studying a whole system). In setting up their lab they explored environmental influences on halo. In order to fit their research into what is understood about halo – they must look at data from other experiments/scientists. Most scientists tend to specialize (and other scientists will be working on related experiments) so they won’t be producing all the data on an organism/system. During the jigsaw each of the 3 data groups will be acting as a separate lab group. When they get together to share their ideas, that will illustrate how scientists work together across many fields to complete the “picture” of a system (e.g. this data would have come from geneticists and biochemists).
Slides 1-6 reviews the influence of light in making bR and reiterates the 2 guiding questions.
Slides 7-12 actual metabolic network is shown.
Slides 13-15 adds in homology data to the metabolic network.
Slides 16-18 shows metabolic network (again) and focuses on the genes and proteins that would be involved in a microarray.
*At this point, students have seen the result of the 3 data sets and can compare to their own understanding. The next slides ask students to make predictions on how various changes in the network will affect the outcome.
Slides 19 - 24 asks ‘which genes are affected by bat’.
*You may wish to point out that enzymes are positively affected (these are the enzymes that become larger in the PowerPoint; when bat is over expressed, these are also over expressed). The effect of when bat is knocked out is then shown. If any enzymes became larger (they actually don’t) they would be ones that are negatively influenced by bat (when bat decreases, the expression of these enzymes increases). This is a good place to cover a Washington state standard on feedback and systems. This concept can be difficult for students, particularly the idea that a correlated decrease in two nodes signifies a positive relationship. For example, the decrease in CrtB1 when bat is knocked out means the relationship between the two is positive.
Slides 25 - 26 offer a quick comparison of knocked out and overexpressed bat (again).
Slides 27 - 30 students are asked to predict how bat affects the amount of bR produced. Click and the metabolism network is added. Click to see this network when bat is knocked out. Click again to show overexpression of bat. Click to see them at the same time. Allow students to study this slide.
Slide 31 reminds students of the 2 guiding questions (see slide 6).
Slide 32 shows the network set up. Ask students to predict what will happen as light is added. Ask them to closely watch the bR and ATP. Click and an arrow goes from light to bat. Click again and arrows go from bat to CrtB1, brp, and bop. Click again to increase bat; click to show that CrtB1, brp, and bop increase and then click and wait as the network increases from GG-PP to ATP.
*Ask students the mechanism of how bat is influenced by light. What is the exact role of bat? Answer: Bat increases transcription of genes associated with the formation of bacteriorhodopsin (handout calls it a Transcription Regulator). When light is present, it is able to bind to the UAS region more efficiently (due to a shape change), increasing transcription of these genes. Due to the cascade of reactions, increasing bop and the enzymes leads to an increase in BR production. There are two slides that will illustrate this point with an animation.
(Homology group students should be best able to answer this question.)
*Make sure you get whole class to understand bats role in transcription of the other genes.
Slide 33 shows the network. Ask students to predict what will happen if light is absent. Click- light fades. Click-arrows show bat influences CrtB1, brp, and bop. Click and bat gets tiny. Click and then so does CrtB1, brp, and bop. Click to watch the nodes in the network change.
Note: See the bR Network Simulation to understand that oxygen does not have the same relationship with bat as light. Oxygen negatively impacts the binding of bat which results in a decrease in BR if oxygen is high.
Slide 34 Point out to students that they have determined the network for producing bacteriorhodopsin. Note the + signs are included to show bat has a positive relationship with CrtB1, brp, and bop in the presence of light.
This slide also asks about other possibilities. Students can hypothesize when they think a fermentation pathway would be “on”. From their research students may remember that this pathway for energy production is used when there is no light (and no oxygen). At least students may bring up the possibility of a network that operates in the dark. *halobacteria can produce ATP using fermentation.
Slides 35-36 shows the bR network in the presence of light. Click to add the fermentation pathway.
This bottom pathway (arginine fermentation) is currently hypothesized to be another way that halobacteria can make ATP. Alternative pathways allow halobacteria to survive in a variety of different environments (extremophile).
Note: this network does not include ATP gained through the aerobic cellular respiration pathway. To add oxygen’s influence on bat and include this pathway would make this diagram much more complex. For simplicity it has been left off (additionally the full interaction of all the pathways and their regulation is still be studied).
*Just know (as simulation shows) - in high oxygen conditions, the light pathway is off since most ATP is being produced by the more efficient aerobic respiration pathway.
Click to remove the metabolites. Click again to show the positive or negative relationship bat has with both networks in the presence of light.
Slides 37-43 the network interacts.
The slides will first show the relationship bat has with certain proteins in the BR pathway AND how this same gene has a negative relationship with proteins in the fermentation pathway. The animation goes on to show when bat is over-expressed (the bacteriorhodopsin pathway is active) and when bat is under-expressed (the arginine fermentation pathway is active). The animation will continue to switch through both bat expressions.
Note: You may need to remind students the difference between a positive and a negative relationship. If bat increases, anything with a positive relationship with bat will also increase, while something with a negative relationship would decrease. The reverse is true if bat decreases (+ would also decrease, - would increase).
Slide 44 asks the class why the cell goes through all the trouble of this complicated network? Why is the bR network regulated? Why doesn’t the cell express all of the proteins at the same time?
This corresponds with the Big Ideas questions you may need to prompt students for them to see how regulated bop would be different than the regulation using bat. This should show students that bat cuts the pathway off at the start, while bop regulation would allow all metabolites to be produced (stops pathway at end). It is wasteful for the cell to produce all proteins all the time. The cell produces energy in the most efficient way. Cells can regulate (turn on or off) genes depending on what proteins are needed by a particular cell. The pathway that halobacteria uses (photo vs. arginine fermentation) depends on the environmental conditions and the most efficient way to produce ATP.
Slide 45 asks students to think about how we could test the model.
Emphasize that the final constructed network is a hypothesis or model of what we think is happening in the network based on the data.
The students should come up with the light/dark experiment that they just did. They should recognize the limitation of this experiment (can’t tell for sure involvement of bat). Students that had microarray may bring up the idea of a mutant organism (one that has over or under-expression of the bat gene). If students don’t get there, that is fine. Basically, the main point of this question is for students to realize that even after looking at all this scientific data, they still would need to do experiments to show how the whole network interacts together in response to environmental changes (i.e. how systems biology is studied).
Bring the class back to the experiment and talk about how this energy network could be used to analyze experiment results. For classes that have not yet collected their data, a good formative assessment could be to ask each lab group to predict the phenotype of their experiment (at this point the answers should just be purple (lots of BR) or less purple/pink – based on the color of the Halo when the students start). They may also be able to predict whether their sample should have had minimal, average or a lot of growth. If asked, feel free to remind students that cells need energy to divide. So cells that have lots of energy (using oxygen to perform cellular respiration) will have lots of growth, followed next by those with light but no oxygen and lastly those samples that should only be performing fermentation (no light AND no oxygen).
- Students’ hand drawn network of the bR network and answers to analysis questions through oral discussion.
- Students’ hypotheses for how the network regulates the production of bR.
Scientific Explanation of how bat oxygen and light modify protein struction and activityHuman Genome Animation from NHGRI
- Microarray data:
- Homology data:
- Metabolic data:
- Overview of regulated transcription, beginning with a stimulus. Please note that this video pertains to a eukaryotic cell with a nucleus and a chemical stimulus. While Halo does not have a nucleus, the overall process and concepts are comparable.
- Another good video is this one from Paul Andersen of Bozeman Biology. He first describes positively and negatively controlled gene expression in E. coli, then at 8:23 addresses gene expression in eukaryotes. Halo process their genes in a way that is similar to eukaryotes by using transcription factors. After showing this video, ask students what makes those transcription factors bind or release? In Halo's case it is the amount of oxygen present. The Bat protein (a transcription factor) contains an iron group. That iron group binds oxygen when in the Fe2+ state, changing it to Fe3+, and causing a conformational change. That change means it can no longer bind DNA. Once it is not attached to the DNA, transcription stops and the genes coding for the production of bacteriorhodopsin are no longer expressed. This makes sense, because the cell does not need as much bacteriorhodopsin when there is a lot of oxygen around. Cells can instead use cellular respiration for their energy needs.
- Here is a zoomed in animation depicting how transcription factors bind. Again, while this is not exactly what is happening with bat in Halo, it does help visualize the process of regulation.