Isolation and characterization of antibiotic-producing soil bacteria

Maria Messner, Lenoir Community College

Location:

Abstract

One of the biggest threats in hospitals is the rising number of patients who harbor antibiotic-resistant bacterial strains. Therefore, it is critical to find and characterize novel antibiotics to combat the resistant strains. Most of the antibiotics used in healthcare settings were isolated from soil-derived antibiotic-producing bacteria and fungi. The research goals are to isolate antibiotic-producing bacteria from local soil samples and to determine the activity and structure of the antibiotic(s). This CURE aims to engage undergraduate students in a process of scientific discovery, iteration, and contribution to the body of knowledge.

Student Goals

  1. Learn basic laboratory, math, reading comprehension, and computer skills.
  2. Develop scientific inquiry skills by performing experiments that aim to answer the research questions.
  3. Learn how to effectively communicate orally and in writing the relevance of their work, the process they have taken to complete the work, and the conclusions they can make.

Research Goals

  1. Isolate novel antibiotic-producing soil microbes using differential media.
  2. Determine the biochemical properties and antibiotic production and efficacy of the microbe of interest.
  3. Identify the antibiotic factor.

Context

This CURE was designed to offer the largely underrepresented and underprepared students at a rural community college an opportunity to engage in real scientific research. The CURE replaced the inquiry-based labs in two-semester General Chemistry I and II courses. Throughout the semester, standard inquiry-based labs (acid-base titrations, chemical kinetics, equilibrium, etc.) were conducted to supplement core Chemistry content, thereby making the courses interdisciplinary. Laboratory sections ran for 3 hours per week, numbered 18 students maximum, and students worked in groups of 3-4. The instructor takes on the roles of lecture and lab instruction and preparation. The first few weeks start with an introduction to basic lab equipment and practice using the equipment, measurements, and information about the project, as well as soil collection and plating. The rest of the semester progresses to an introduction to peer review, culturing soil bacteria on differential media, and assessment of biochemical activity. Students use peer-reviewed journal articles as templates to write the materials and methods, introduction, and results.

Target Audience: Freshmen and Sophomores
CURE Duration: Two 16-week semesters

CURE Design

Discovering new antibiotics is the theme of this CURE. The project is designed to expose students to learn basic scientific skills (measurements, lab safety, etc. ), learn project-related lab techniques (sterile technique, centrifugation, subculturing, serial dilutions, PCR, etc.), gain biological and chemical knowledge (distinguishing features of prokaryotes and eukaryotes, ways cells communicate internally and externally, how hydrogen bonds affect thermocycler parameters, the importance of buffers, etc.), and to learn the process of scientific inquiry (questions, discovery, iteration, discussion, etc.). The learning environment is structured to ensure all students are successful in achieving the goals of the CURE by communicating clearly the goals, objectives, assignments, and rubrics, as well as giving individual attention and encouragement.

Most of the antibiotics in use today have been isolated from soil-derived, decomposer bacteria, most notably Streptomyces spp, which also were discovered to produce antibiotics. Students collect soil samples from their chosen GPS coordinates. Samples are dried, serially diluted, and cultured onto Actinomyces Isolation Agar. Students will select a colony following subculture to test for possible bactericidal and bacteriostatic properties via perpendicular coculture. Colonies are processed for 16S PCR amplification for sequencing and subsequent BLAST for bacteria identification. Colonies are characterized for biochemical activities using differential media. Chemical analysis of antibiotically active colonies is conducted using GC-MS. Novel antibiotics will undergo further structural analysis and characterization. Students are to develop a hypothesis and design an experiment to test the hypothesis regardless of being able to discover an antibiotic-producing colony.

Core Competencies: Asking questions, Planning and carrying out investigations, Analyzing and interpreting data, Obtaining, evaluating, and communicating information
Nature of Research: Basic wet lab research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Isolate soil bacteria (Streptomyces spp), identify the species, and determine biochemical properties.
Research Goal 2: Determine the efficacy and identity of the antibiotic factor.


Student Goal 1: Learn basic lab techniques, and reinforce math, reading comprehension, and computer skills.

Conduct a library database search on and summarize current knowledge of antibiotic-producing bacteria.
Identify the functional parts of a scientific journal article.
Produce a data table graph from sample data.
Practice sterile technique.
Correctly pour agar.
Maintain a laboratory notebook based on established guidelines.

Summarize the main molecular features and mode of action of known antibiotics.
Learn the functional groups of organic compounds.



Student Goal 2: Develop scientific inquiry skills.

Observe and record colony morphology on a plate.
Learn and identify the biochemical properties of the bacteria by choosing differential media.
Troubleshoot a failed experiment and determine solutions.

Learn about and practice lipid extraction.
Learn about and run PCR.
Extract antibiotics using organic solvents.
Correctly run thin liquid chromatography plates.
Use an online database to compare PCR data to documented known strains.
Develop ideas for future experiments.



Student Goal 3: Learn how to effectively communicate orally and in writing the relevance of their work, the process they have taken to complete the work, and the conclusions they can make.

Engage in group discussions about the project in lab meetings.
Write the methods and results using peer-reviewed publications as a template.
Participate in blind peer-review of the methods and results.
Make a poster and present it.

Write a research proposal based on example proposals.
Participate in blind peer-review of the proposals.
Give a PPT presentation on their research proposal.
Make a poster and present it.


Instructional Materials

Aseptic technique and isolation Fall 2018.docx (Microsoft Word 2007 (.docx) 52kB Sep24 19) 
Agarose Gel Electrophoresis Fall 2018.docx (Microsoft Word 2007 (.docx) 21kB Sep24 19)

Colony PCR Protocol FAll 2018.docx (Microsoft Word 2007 (.docx) 20kB Sep24 19)

How to write a research proposal.docx (Microsoft Word 2007 (.docx) 20kB Sep24 19)

Assessment

Lab notebook rubric (Excel 2007 (.xlsx) 12kB Mar5 19)
Rubric for Project Proposals.docx (Microsoft Word 2007 (.docx) 18kB Sep24 19)

Instructional Staffing

The instructor prepares media, solutions, and maintains equipment, and provides group instruction and guidance, and one-on-one mentoring.

Author Experience

Maria Messner, Lenoir Community College

My motivation to develop and implement a CURE is rooted in a desire to train students to gain the practical and cognitive skills and knowledge to do well in their chosen fields. CUREs have been proven to be effective in increasing student retention and excitement in STEM fields, and in improving writing, presentation, and critical thinking skills.

Advice for Implementation

This CURE was adapted from the Freshman Research Initiative at the University of Texas at Austin. They shared many resources that made it possible to start. The resources can, and will likely need to, be tailored. Know the level at which students are entering, and the number of hours they are scheduled to be in the lab. Be prepared for the project to move at a slower pace than anticipated. Depending on the course level, build in time for students to practice techniques, and for review and instruction for basic math and laboratory techniques. Make use of pre-lab videos that show lab techniques and conceptual ideas. Pre-lab quizzes help motivate students to watch the videos. Help students organize their bench space and provide examples regarding how to work through a protocol. For example, during PCR prep, students must add various components to their tubes and may not remember what component has been added to which tube. Labeling tubes and moving the tubes to a different row or part of the ice bucket will help. Making use of checklists will help them to remember to add tips to micropipettes, have ice ready, etc.). Remind the students periodically that science is an iterative process and that troubleshooting is part of it.

Iteration

Students learn about the parts of a manuscript and use primary journal articles as templates to write those sections. Assignments require improvements on drafts as they first receive peer review, then instructor feedback. Experimental iteration is common as they are learning many new techniques and must troubleshoot.

Using CURE Data

Data may reveal the characterization of novel antibiotics.

Resources

These resources are used to show students scientific writing style (third-person passive voice), to identify the parts of a peer-reviewed article, and to serve as a template for writing their materials and methods. They are not expected to understand all of it.

Bundale, Sunita et al. "Culturable rare actinomycetes from Indian forest soils: Molecular and physicochemical screening for biosynthetic genes." Iranian journal of microbiology vol. 10,2 (2018): 132-142.

Lee, Learn-Han et al. "Diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in Malaysia." TheScientificWorldJournal vol. 2014 (2014): 698178.