Lesson 2 – Response of Halo in Different Environmental Conditions

Before beginning lesson: Read entire lesson. The following documents also may help with prepping for this lab:

Students reviewed literature about a model organism (Halo) during Lesson 1 which gave the students an opportunity to investigate the environmental conditions Halo typically grow in. The Lesson 2 laboratory experience will give students an opportunity to actually see the effect of certain environmental variables on the Halobacterium organism. Depending on the time constraints and goals for this lesson – there are 3 possible pathways. Here are the possibilities:

1.    Students work with a defined manipulated variable – presence of light – AND a defined, predetermined lab protocol. NOTE – This will remove one 50 minute period from the time required for this lesson, however it will be harder to present as inquiry: observations lead to hypotheses and experimental design.

2.    Students work with a defined manipulated variable – presence of light – they will design a laboratory experiment using this manipulated variable which then can be carried out in the laboratory. Teacher will need to guide students with the availability of their laboratory supplies (use of stir stations, agitating incubator, bubbler system).

3.    Students work with different manipulated variables that are determined during their Lesson 1 research. The possible variables that can be easily manipulated using the kit (provided you have enough lab equipment) are: light, oxygen, salinity, color of light, pH, temperature, amino acid concentration, presence of metals or oil.

Research Reflection:

Variable Selection:

• This can be done by giving student groups whiteboards or a planning format to help them get organized.

• Remind students that only variables that make sense should be used (i.e. no aspirin, soda, etc.).
• If time – students should brainstorm lab options (i.e. equipment needed or how would the variables be modified or measured).

• Environmental Ideals – Temp (~37C to 42C – 37C is suggested), pH (neutral), Salinity (3-4M)
• Ecology of Halo – Definition of an Extremophile – Extremophiles are organisms that live in harsh environments and are members of the Archaea domain. Note: this domain is not part of the true bacteria (eubacteria) domain but are made of prokaryotic cells (like eubacteria)
• bR causes the color of Halo (this is a purple pigment). It is involved in the energy pathway that uses light.  “Light” in this case is full spectrum light.  See the teacher resource, “Advanced Prep Document” for more information on what types of lights can be used.  Specifically, bacteriorhodopsin (bR) is most efficient at absorbing green light.
• Methods for creating usable energy – 3 different pathways – use of light (phototropic); use of oxygen and organic molecules (cellular respiration); anaerobic metabolism of arginine (fermentation)

• light, oxygen, salinity, color of light, pH, temperature, amino acid concentration, presence of pollutants (like metals or oil), carbon dioxide (this last one can lead to a conversation on whether we think this could have an effect – since they don’t use the CO2 for energy although it can have an effect on pH of the system).

• NOTE: If your students have no experience with the spectrophotometer – you may want to complete Lesson 5 in the Ecological Networks module to assist with the workings of the spec.
• If your students are designing their experiment, teachers can use the following prompts to lead students to valid design.
  • Will this give you accurate results? – Students should always run three sample flasks or tubes, plus a control to be a statistically viable test.
  • Will your Halo be able to survive those conditions? Is it useful if your Halo all die in the experimental groups?
  • Why did you pick those levels of your independent/manipulated variable?
  • What is your control group?
  • Does that variable have any other impact on the environment? – e.g. temp can change pH

• Remind students that a goal of this module is to figure out why and how the environment influences gene expression. Tell students that while each group has a feasible experiment, the light variable is not only quite easy to control in a basic lab, but also, the gene network impacted by light and oxygen is one that we already have much data on. This means that we can combine our class data with a great deal of known data to really have a shot at answering our questions and understanding this network. Since this is our first full network analysis, it would be a good place to start and then would allow us to have more success when studying less known networks in the future.
• The protocol for the lab can then be handed out (Testing light on Halo with stirrers-STUDENT (Google Doc | Word Doc), Testing light on Halo with incubators_STUDENT) (Google Doc | Word Doc). The students can record their data in their lab notebooks using this format: Testing light on halo-STUDENT DATA SHEET (Google Doc | Word Doc) or STUDENT RAW DATA SHEET  (Excel)
• There is also a document that describes what you need to know for this protocol:
  • Testing light on halo-TEACHER (Google Doc | Word Doc)
  • If you do not have stir stations, shaking incubator or like device, you could adapt this protocol as needed to test just light and/or oxygen also. Here are documents to guide you:
    • Intro document: Aerator Experiment Intro with Pre- and Post-lab questions (Google Doc | Word Doc)
    • Lab investigation: Aerobic Anaerobic Light Dark Experiment with Aerator (Google Doc | Word Doc)
• Keep in mind that when testing aerobic and anaerobic samples, your “anaerobic” sample is not truly anaerobic unless you have flushed all of the oxygen out with some suitable gas. Even though most schools do not have this capability, the sample is nearly anaerobic since, oxygen does not dissolve readily in the high salinity media for Halo. Typically, only the cells growing on the very top layer of the media in the flask have access to oxygen. Given the overall volume of the sample, this should give clear results but would be a good thing to discuss with students.

• Once students have come up with a list of variables, teacher than picks light as the manipulated variable for the class (see note above in maroon). Students will now need to design how they will modify the light in the laboratory section. During this section students can work to design in their lab groups. Teacher will need to show students the supplies available for them to use during the experiment. In the end, teacher should help guide students to a valid protocol. This may take some time depending on how much experience your students have with designing labs. Students will most likely need additional information from you to be successful in this effort. This document (Student Lab Hints) (Google Doc | Word Doc) was created as a background protocol handout to give to students. You should decide when would be the best time to give it to your students in the design process.

• Once students have come up with a list of variables, teacher will need to help students eliminate those variables that cannot be testing with their laboratory supplies. Once the list is finalized, each group needs to select a variable from that list. The lab groups should then begin to complete their lab experimental design. Each groups should receive Student Lab Hints  (Google Doc | Word Doc) which will give the students information/hints for their protocol design. This process is probably best to be done initially on a white board (which will allow the teacher an easier method for approval).
• Once students have a valid experimental protocol – they can complete a pre-lab  with a rough outline of their procedure (see below note).

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To review experimental design and thinking about variables, ask students to answer a question on plant mass.  Hand out the  question, collect answers and tally on the board, read aloud, or have students answer anonymously and then crumple their paper and toss to the other side of room. Students pick up and read another student’s answer.  *Group students (human histogram) according to answer.  Likely answers are: light, water, air, soil, fertilizer.   To investigate the answer, use a projector to display and explore the following web-page: