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.
Students should refer to their notes from their Halobacterium research. Students should be put in lab groups (3-5 students per group). They should then spend no more than 10 minutes comparing their research results to be sure the entire group has everyone’s information. This will also allow them to see that different sources may have slightly different information (e.g. ideal salinity or temperature). Students may have some holes in their research that group members can fill – any new information should be recorded.
After the information share, groups should come up with a list of possible, valid independent/manipulated variables that could be tested in the school’s laboratory (at least 4). Student groups should also come up with possible responding/dependent variables (at least 2).
· 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).
Class Discussion of Research:
This discussion should end when all important/relevant information has been gathered from the students. Students should also be sure this information is recorded. This may include the following:
· 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)
Determine possible variables for Experimental Design:
Lead students through discussion on which variables could be tested with Halobacterium. NOTE: Depending on your plan for the lab, this can be limited to what they can do in your schools laboratory or wide open. If you allow any valid variable to be discussed, you could continue the discussion on HOW each of these would be tested (which may eliminate some choices from your list). Here are a few of the possible independent/manipulated variables you may get:
· 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).
The class will also need to talk about what dependant/responding variables could be measured. Possible list: population (growth rate, amount of growth, size of population, survival rate, density, pattern of growth), color, organism size, behavior. Consider constraints due to lab equipment available.
Experimental Design – Using Halo to test how the environment can change population size and gene expression:
This step is determined by which path you plan to take for the lab.
· 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.
o Will this give you accurate results? – Students should always run three sample flasks or tubes, plus a control to be a statistically viable test.
o Will your Halo be able to survive those conditions? Is it useful if your Halo all die in the experimental groups?
o Why did you pick those levels of your independent/manipulated variable?
o What is your control group?
o Does that variable have any other impact on the environment? – e.g. temp can change pH
1. Students working with light AND defined lab protocol:
Once students have come up with a list of variables, teacher then picks light as the manipulated variable for the class.
· 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.
· 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.
2. Students working with light but designing lab protocol:
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.
3. Students working with variety of variables:
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).
NOTE: Teacher’s access to equipment may limit the student’s flexibility with manipulated/independent variable levels (e.g. the kit comes with predefined salinity levels). As long as students recognize some of their steps may slightly change, this should be fine. Teacher can also describe how this models real scientific lab work where everyone must be aware of their lab’s limitations.
Complete Lab – Using Halo to test how the environment can change population size and gene expression:
Students should now complete a lab according to the protocol they have been given or designed. Please be sure to review the document of laboratory hints/suggestions (Teacher Lab Hints-variables) (Google Doc
| Word Doc
) Even if you are performing the defined protocol, this document may have information to assist in the success of your students’ experiments.
Due to incubation requirements, final data will be taken at the beginning of Lesson 4. If you have equipment limitations which will only allow 1 or 2 groups to work at a time, a different timing schedule will need to be used. See the Advanced Prep document
(at the beginning of module) for suggestions.
You completed Instructional Activities. Please move to assessment