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Life Science and Biotechnology Laboratory Research

Example: Optimizing Plasmid Transformation of Bacteria

(Modified from Laboratory 5: Rapid Colony Transformation of E. coli with Plasmid DNA, in Micklos and Freyer, DNA Science, Cold Spring Harbor Laboratories/Carolina Biological Supply).

By Toby Mogollon Horn



Type of Entry:

  • Project

Type of Activity:

  • Hands-on
  • Inquiry lab
  • Authentic assessment
  • Group/cooperative learning

Target Audience:

  • Life Science
  • Biology
  • Advanced/AP Biology
  • Genetics / Biotechnology

Notes to Teacher:

The process of extended laboratory research helps students experience how scientific information is obtained. A variety of scientific methods yield the information upon which we build scientific knowledge and understanding. Among the methods in the scientific inquiry repertoire, researchers may use surveys, observations, manipulations, theoretical models, or simulations. In the course of a scientific research project, students may use one or more of these approaches. The results may not be perfect, so students gain experience in working with real-life data. In the example described, students develop a method by using survey and manipulation approaches.

Senior Life Science and Biotechnology Laboratory Research is an 8-year old, on-going, one semester, 90 minute work period that fulfills a graduation requirement in technology. All science students need direct experience connecting the original discoveries made by scientists during the experimental process with what they perceive as "science facts" in a textbook or news report. From combinations of invisible chemicals, tiny cells and "black-box" instruments, high school students working like scientists generate notes, numbers and images that they themselves transform into tables and graphs and then interpret, redraw and reexamine experimentally. Each day can provide a new discovery, many mistakes, and, perhaps, an answer; each work time -- more questions. Among the many ways to do research, a few features provide a framework. *Pose a simple yet thoughtful question. *Learn how to handle your "system." *Work at the answer with care. *Stay open to new questions that might be easier to answer.

Based on my own experience at the bench, I provide students with an in-school mentorship that models my own research training. Not only reasoning and math, but also handiwork skills, detective work and perseverance contribute to success in a research project; all these need conceptual and factual background, methodological discipline, AND imagination. I view the Biotech Lab workplace as a studio filled with young Michelangelos who I encourage (and sometimes cajole) to flower through daily work in the scientific realm. Few students have used imagination in science before.

Though all of my students are highly selected (only one in 6 applicants is accepted to our school), within this high range are students with widely varying abilities, interests and motivations, including officially "LD" students for a variety of processing and other aptitudes. Since I work with each one (of a class of 15) individually, during class I can find each student's strengths. The students use similar processes at different times so each can eventually teach another, which is especially empowering for a student who starts at a low ability level in some facet of the work.

An idea that piques a student's interest is the starting point for each research path. Many a student comes to the lab with "great interest but no idea of what problem to study." I ask each to THINK about some reading for another class or memorable news or scientific report. Usually the first or second thing that comes to mind is something that was neat to learn about OR difficult to comprehend. We scientists like to study what is difficult to understand and explore the unknown. Our imaginations lead to creative solutions. We seek out problems because they are serious fun to study; problems are not things to avoid but opportunities to learn.

By the end of the semester project time, most students will have collected data to produce an interpretable result. During the process, each student searches the scientific literature and the Internet, negotiates a project that is similar to (or models) the question of interest based on time, safety and equipment that's available, defines a topic and title, writes a research proposal, prepares chemical solutions, culture media and working materials, maintains and/or characterizes the organism or biochemical of interest (most projects involve a cellular process or function), learns to use several different kinds of tools and instruments, collects basal and experimental data and writes a final report. Most students find an email mentor expert in their field -- which helps ME out a lot as well.

All students are initially responsible for "soup to nuts." Then they must teach me how to prepare their materials and leave comprehensive instructions. All succeed in some aspect of their work. Each develops an appreciation for what went into a paper's charts or tables. Most gain healthy skepticism. Though some students begin frustrated or angry " "cause it didn't work the very first time" or I don't just give them the answer (I don't know the answer, of course!) and many end up with negative results, all leave Biotech with a sense of real accomplishment.

Teachers can introduce students to independent laboratory research project work by focusing on a topic with which the teacher has some methodological familiarity. During the first year of developing a program for independent research projects, I devised a 1-3 week unit during which teams of students examined various aspects of one type of experiment and compared their results. In this example, the rapid colony transformation procedure is first performed, then extended to determine if various parameters are maximized, optimized or minimized. With a large class, more than one team of 3 students can examine each parameter and these teams can compare their results to assess reproducibility among groups.

Features of a process-oriented laboratory research project are:

  • an answer can be obtained whether or not it fits the student's hypothesis

  • students can become skilled at the methods in a reasonable time

  • the baseline, standard and controls can be clearly examined

  • data are of a type on which numerical or scoring analysis can be done

  • time is provided for discussion of results, refining the experiment based on the discussion and conducting the refined procedure.

Based on research by several groups of investigators, parameters have been optimized so that colony transformation can be conducted during a high school lab period. How did this method come to be? What role does each reagent serve? Using pAMP plasmid and MM294 cells, perform the rapid colony transformation procedure as described in Micklos and Freyer and analyze the results for all class teams. Then, each team examines one of these: choice of divalent ion (try magnesium or manganese chloride), varying calcium chloride (divalent cation) concentration, time or temperature of heat shock, concentration of plasmid, length of incubation of plasmid and cells, or length of recovery.

Required of students:

Cooperate so the whole team is successful intellectually and methodologically, take good notes. Each student is responsible for proper handling and maintenance of a piece of equipment or biological material or reagent; working out alternate ways to analyze the data; discussing the results and brainstorming refinements.

Preparation time needed:

Biologicals, culture plates, and stock solutions. Later, students prepare all working materials. Recipes and ordering information: Micklos, D. A., and G. A. Freyer. 1990. DNA Science, Carolina Biological Supply Co., Burlington, NC.

Class time needed:

2-3 weeks to prepare materials, conduct the experiment twice, discuss and refine, and conduct the experiment again.

Additional References:

*Mandel, M., and A. Higa. 1970. Calcium-dependent bacteriophage DNA infection. Journal of Molecular Biology 53:159-162.

*Hanahan, D. 1985. Techniques for Transformation of E. coli, pp.109-136. in D. Rickwood and B.D. Hames, eds. DNA cloning: A practical Approach. Vol. 1. IRL Press, Washington, DC.

*Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

*Cothron, Giese and Rezba. 1993. Students and Research. 2nd ed., Kendall Hunt 4050 Westmark Dr Box 1840 Dubuque Iowa 52004-1840

*Rezba, Sprague, Fiel and Funk. 1994. Learning and Assessing Science Process Skills. Kendall-Hunt, Dubuque, IA.


Project

Life Science and Biotechnology Laboratory Research Optimizing Bacterial Transformation by Plasmid

Materials

All materials are available from Carolina Biological Supply as listed in Micklos and Freyer, DNA Science. These include: pAMP plasmid, MM294 cells, LB agar, calcium chloride stock solution, ampicillin antibiotic, pipettors, pipettes, spreaders, disinfectant, biohazard bags.

Equipment:

*Autoclave or pressure cooker for sterilization during preparation and disposal phases

*Waterbaths

*Incubator

*Markers

Alternate sources include:

Life Technologies Inc., for LB agar powder, ampicillin

*Baxter/SP for Petri Plates, pipettes, pipettors and Calcium Chloride dihydrate powder, waterbaths, insulated containers

*Waterbaths can be styrofoam insulated containers or ice buckets (insulated foam) with an appropriate mix of hot and cold water to obtain the desired temperature.

Students should first conduct the rapid colony transformation lab (Laboratory 5 in DNA Science) as a regular class lab. This method was worked out by several groups of professional researchers who used a variety of experimental approaches, surveying different materials, examining the functional activity of certain reagents and conducting kinetic and concentration studies.

The transformation procedure requires the following components:

  • Plasmid DNA solution
  • Host bacteria suspension
  • Calcium chloride solution
  • Icebaths and Waterbaths
  • Bleach
  • Recovery liquid medium
  • Petri plates with selective medium
  • Bunsen burners
  • Spreaders
  • Pipettes
  • Pipettors
  • markers
  • 15 ml tubes
  • Inocultation loops

In this method, many parameters are specified: volume and concentration of plasmid DNA, bacteria in a particular physiological state, divalent cation at a particular concentration, incubation on ice for a specified time, heat shock at a certain temperature and for a specified time, recovery for a designated time.

Each of these specifications is based on a series of experiments to define and refine conditions to get the best results in the shortest time most easily. How and why?

Each team will test the effect of varying one component, keeping all other factors constant. Results will be reported as number of colonies on selective plates (dependent variable) vs time, temperature, concentration, type (independent variables, see A-G, below). Conduct your experiment in duplicate and repeat the experiment before preparing your initial report for presentation and discussion.

A. amount of plasmid DNA
B. type of divalent cation (Mg, Mn, Ca all in Chloride form)
C. concentration of divalent cation
D. incubation time on ice
E. heat shock time
F. heat shock temperature
G. recovery time

Instruct students to consider each of these aspects of conducting the laboratory work.

Brainstorm what levels of variable you want to test. Be certain to include the standard condition and a blank (0 time if you are conducting a time study or 0 concentration if you are adding something).

Set up a chart to indicate each substance to be added and/or time so you can check off as you complete each addition or process. Test/assay each variable and blank in duplicate. Repeat the procedure after you obtain and analyze the first set of results.

Plan out how much of each component you will need, including cells, so that you can prepare the appropriate number of starting plates, have a small excess of each solution, etc. Be responsible and cooperative and share excess materials with other teams. Schedule use of water baths and other scarce equipment with other teams.

Prepare enough plates, solutions, reagents: Prepare at least twice the needed amount per experiment.

Remember to test your standard each time and test only one or two variables until the team works up to examining more variables at one time.

Delegate tasks so that each team member is contributing to the lab work. Have each member add a reagent in turn. This is a good way to ensure that everyone gets to pipette and it also saves time since a second addition can be started as each tube has received the first item.
During the repeat of the process after discussion and refinement, students can jigsaw so they can contribute their expertise to a new group.

Analysis of results

Obtain the averages of the duplicate assay tubes and plot your results as a graph with X and Y axis of equal length. Plot the variable you manipulated (time, temperature, concentration) on the X axis and the result (colony number) on the Y axis.

If more than one group examined the same parameter, show your results side-by-side or on the same chart with each group using a different symbol.

Literature search and analysis

Using your library resources or a local college or the internet, find out what other work has been done in this area. For example, look up how divalent cations affect DNA uptake. Scientists adapted procedures for working with other types of DNA in developing the transformation method described in Micklos and Freyer.

Compare and contrast the method in Micklos and Freyer with procedures described in other professional manuals.


Method of Assessment/Evaluation

*Participation in group work

*Planning

*Progress

*Lab book documentation

*Experimental design

e.g.: Are the duplicates clear? Is the standard included? Is there a control or blank? Do students consider how to compensate for differing cell concentrations in each colony. ( Make one suspension containing many individual colonies when testing several variables of a parameter.)

*Description of rationale and methods

*Graphical presentation and description

*Oral presentation

*Relevant references and reference report


Extension/Reinforcement/Additional Ideas

A. Characterizations of enzyme activity. Several inexpensive proteases are available. Teams can each analyze one enzyme. Parameters to study include temperature, type of substrate, substrate and enzyme concentrations, pH, detergent resistance. Controls include blanks without enzyme (especially important in acid pH). Students graph results and can also figure out the specific activity of the enzyme by conducting a protein assay.

B. Heat resistance of Bakers' Yeast. Teams use fresh proofed yeast or stationary or log phase cultures, and make water baths for short term incubations at temperatures from 0C to boiling. Methyl blue dye metabolism distinguishes live and dead cells. Viability of wet mounts can be quantitated using a microscope, if possible using a hemocytometer. The data can be entered into a spreadsheet such as Clarisworks or Microsoft Works or Excel for graphical and statistical analysis.


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