Lesson 3 – Data Analysis to Propose Network Function

Description: 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.

Objectives

Course:  Biology, Genetics, Biotechnology, Environmental Science

Unit:  Genetics and Heredity

See the NGSS listed in buttons on the upper-left of this page. Also, see the Standards Addressed page for more information and all NGSS and WA State Standards (Science, Math and Literacy) addressed in this module. In addition to the aligned standards, for this lesson, here is a breakdown of:

What Students Learn:
  • 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.
What Students Do:
  • Students analyze three different data sets to determine the bacteriorhodopsin network and to determine the importance of compiling multiple sets of data.

Instructions

Note: This lesson should be done while the Lesson 2 samples are incubating. Students will get their data for Lesson 2 at the beginning of Lesson 4 (unless your equipment limitations require a different schedule – see Advanced Prep for Module document for timing).

Introduction:
Purposes of each element in the lesson
Introductory PowerPoint: Background and review to provide context. Students don’t need to memorize details and do not need to be tested later, but they DO need to understand enough of the context to make sense of the data. A mode of providing the info other than PowerPoint (Google Doc | Word Doc) or material for students to reference may be better.  (Printable script/description) (Google Doc | Word Doc)
Jigsaw Data Analysis: Students will jigsaw the three data sets (Homology, Gene Expression/Microarray and Metabolism) and when completed will have created a model for how the protein Bacteriorhodopsin is created and regulated in Halo using light.  (See ‘Extension’ for an activity involving microarray interpretation.)  See ‘Resources’ for additional teacher information.
 
Class Discussion of Jigsaw: Teacher will lead discussion on the answer to overarching questions:
  • How do Halobacterium cells control the amount of BR expressed in response to light?
  • What is the gene and protein network that regulates the expression of BR?
Teacher should also focus on how this network is a well-understood example of how an environmental change can set off a cascade of events which can change the phenotype (in this case color) of an organism.
Instructions:

Cellular networks and systems biology have been pre-assessed, if your students need some support in this area – it can be inserted here.

Students may need to review enzyme-substrate information prior to this lesson. This is the point where you can review network interactions/analysis with your students if your students struggled with this on the module pre-assessment. If you have not yet covered enzymes, this could also be inserted here.

  • 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.
The PowerPoint (Connecting bR lab data to other data) (Google Slides | PowerPoint) leads a discussion on reasons for this lesson (looking at additional data) and how that data can help explain what students will need to consider when analyzing their data.  (Printable script) (Google Doc | Word Doc).

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 reminds 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).

Jigsaw Groups
Explain that in order to understand how halobacteria regulates the production of bR, various data must be obtained, analyzed and combined to get a complete picture of how the network functions. This is the way modern science functions. Tell students that each person will be responsible for analyzing one of three sets of data and they will then share their findings with a small group. Emphasize to students that there may be names of molecules or genes that they are not familiar with. They will not have to memorize the names of these. The teacher may want to give a brief explanation of naming genes (the names could be anything…).
Divide your class into teams of three (the number of groups depends on the number of students in your class). Assign each student in the team as responsible for a different set of data: homology, microarray (gene expression), or metabolism.
You can either have members of the team work individually on their data sets and questions or you could have members split up and meet with other students who are responsible for the same set of data (groups of 2-3 may be appropriate).  It may be helpful for students to work with others analyzing the same data to discuss the data and the questions.
Each group receives three documents, background information about their technology, the data set obtained from their technology, and the student worksheet for each technology. Students should work in pencil as they might need to make changes to their work.
       *See ‘Resources’ at the end for condensed printables.
Microarray data:
1.      Microarray Background information (Google Doc | Word Doc)
2.      Microarray Data (Google Doc | Word Doc)
3.      Microarray Student Sheet (Google Doc | Word Doc)
4.      Key for microarray (Google Doc | Word Doc)
Homology data:
1.      Homology Background information (Google Doc | Word Doc)
2.      Homology Data (Google Doc | Word Doc)
3.      Homology Student Sheet (Google Doc | Word Doc)
4.      Key for homology (Google Doc | Word Doc)
Metabolic data:
1.      Metabolic Background information (Google Doc | Word Doc)
2.      Metabolic data (Google Doc | Word Doc)
3.      Metabolic Student Sheet (Google Doc | Word Doc)
4.      Key for metabolism (Google Doc | Word Doc)
Students should work on completing the worksheet for ~15-20 minutes. Once students are finished, they should return to their original group. They may need to help others in their group if they are still working.  Once they return to their original team, have them share their information. Give each group the Group Instruction Handout to complete together.  (Group handout answer key.)
Common errors:
a.    Check edge used for BOP + Retinal -> BR. Students tend to write two arrows that both lead to BR. This is not really a correct representation of the relationship, since removal of either product will halt BR production. Be sure students understand the difference between the edge used in the PPT (two arrows into 1) and two separate arrows both leading to BR.
b.    Some students may have put their enzymes in nodes. Feel free to refer them back to their Homology worksheet which does not use nodes. Those enzymes describe the edge, they are not a step the reactant “moves through” in the process of becoming the product. They also are not things we are linking together to show a relationship.
c.  Be certain to go over question 5 from the Group Instruction Handout.  Generally, students will want to give an answer that just reads through the network stating that one node turns into another.  This does not quite answer the question of HOW this happens.  Question them further as needed to get them thinking about HOW this actually happens.  How does an environmental stimulus act upon a cell to get gene expression to occur?  Read the answer key for more information. Specifically, it is all about structure and function.  The presence of oxygen or light changes the shape of a protein, therefore changes its function.  Sometimes that means a protein, or transcription regulator, that could bind to DNA before to begin the process of transcription, is no longer able to (or vice versa).  It is a big “ah ha” moment for students to begin seeing proteins and molecules as things that change shape and activity and bump up against each other to either react or not react.  This connection to structure and function is key to understanding much of biology.  Click here is a more scientific explanation of this process in relation to the the bat and bop proteins discussed in this lesson and in lesson 4. See the accomodations section below for more help on ensuring understanding.
Class Discussion
 After students have completed the network and answered the questions, use the BR network group debrief PowerPoint to scaffold the summative discussion. This shows the network as it should be drawn by the students, showing the information that is added piece by piece. Play the slideshow or some animation will not work. Script for ppt with details:  (printable script)(Google Doc | Word Doc).

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).

You completed Instructional Activities. Please move to the next tab above: Assessment.

Assessment

How will I know they know?

  • 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.

 

Resources

Accomodations

Students who struggle with scientific vocabulary may experience difficulty with the data comparison jigsaw.  Assign data set and group students so that each student’s strength is utilized.  ‘Microarray data’ is the most vocabulary challenging, ‘Homology data’ requires organizational skills and vocabulary, and ‘Metabolism data’ while dealing with biochemistry is the least vocabulary challenging and the simplest to decipher.
Students may benefit from powerpoint slides being printed as handouts with lines for notes.
As stated above in bullet 9c, students may have a difficult time understanding the physical nature of the environment interacting with molecules and regulating the expression of genes. You can assess student understanding of this area specifically by reading their answers from #5 in the group worksheet.  If students need more help, have them kinesthetically act out the process of a cellular stimulus leading to a structural change which leads to a functional change.  One student can be a photon, one can be oxygen.  Others can be transcription factors, DNA (of various forms – genes, promotors, TATA box, etc.), RNA Polymerase, ATP Synthase, and so on. Students can be broken into teams to practice and then perform the act of a stimulus causing a conformational change that results in the binding, or releasing, of a transcription factor that begins the process of transcription (or the pumping of protons, etc.).  Or this can even be done as a whole class activity, complete with student-made props, while they work together to act out the various phases of this process.  The purple membrane simulation in Lesson 4 will also help students understand all the parts working together in this well orchestrated and dynamic system.  Also, these video links may further help students visualize the process of regulation:
  • 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.

 

Extensions

A ‘wet-lab’ simulation to show students how microarrays work, http://www.bio.davidson.edu/projects/gcat/HSChips/Chip_Simulation.pdf led to development of a kit from Carolina Biological complete with a Teacher Guide and Animation.  Thanks go to A. Malcolm Campbell and colleagues from Davidson College.

 

A ‘dry-lab’ simulation to show how microarrays work.  Teacher guide,  student information, student data. (Thanks go to Seattle BioMed’s education program director, Theresa Britschgi and to EMBL.)

 

Scientists expand their understanding of gene expression by ‘knocking out’ a gene to determine how it affects a network.  By removing a gene (sap102) thought to be involved in learning and memory, scientists determined that it is indeed, active.  Students are given some background information by way of video and animation and then can see the experiment used and data collected to support this conclusion. (Adapted materials from Dolan DNA Learning Center at Cold Spring Harbor Laboratory.)

 

Scientists compare DNA or amino acid sequences of organisms to determine how closely related they are.  This site is a good, quick review of homology and bioinformatics involving the same model organisms seen in the previous extension.  (Adapted materials from Dolan DNA Learning Center at Cold Spring Harbor Laboratory)

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