DNA cloning and protein analysis of animal-heme peroxidase within collagen IV of the extracellular matrix

Isi Ero-Tolliver, Hampton University

Location: Virginia

Abstract

This CURE is to expose undergraduate students to the process of DNA cloning to identify the critical amino acids of the animal-heme peroxidase,peroxidasin, responsible for catalyzing sulfilimine bond formation within collagen IV of the basement membrane. Students will bioengineer a variety of mutants through primer design and polymerase chain reactions that contain point mutations within the immunoglobulin domain of the peroxidasin.

Student Goals

  1. Analyze, annotate scientific literature, identify the void in background literature
  2. Design a research project that is hypothesis-driven and builds upon the previous knowledge about animal-heme peroxidases, peroxidasin
  3. Communicate their research ideas orally and through writing

Research Goals

  1. Identify and characterize the role of Peroxidasin within the extracellular matrix by creating mutants within the PxdIg domain using point-directed mutagenesis
  2. Analyze protein expression of the mutants within the extracellular matrix

Context

The CURE is designed for a student group of 20-25 freshmen or Sophomore students. The time needed is at least one semester per research goal. The students need to know how to read scientific literature, pipet, how to do calculations and conversions, solution preparation and dilution, and possibly cell culture.

Target Audience: Introductory, Major, Upper Division
CURE Duration: A full term, Multiple terms

CURE Design

The theme is participation in student-driven, hypothesis-testing research on cloning through point-directed mutagenesis and protein expression

Core Competencies:Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Constructing explanations (for science) and designing solutions (for engineering), Planning and carrying out investigations
Nature of Research:Basic Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Identify and characterize the role of Peroxidasin within the extracellular matrix by creating mutants within the PxdIg domain using point-directed mutagenesis
Research Goal 2: Analyze protein expression of the mutants within the extracellular matrix


Student Goal 1: Analyze, annotate scientific literature, identify the void in background literature

Attain 5 current literature articles on peroxidases from database including Pubmed.
Comprehend and summarize the current literature on peroxidases and Peroxidasin.
Attain and read the mutagenesis manual for protocols and alternative approaches and trouble shooting techniques.
Complete an online quiz on mutagenesis manual.
Articulate or develop a flow diagram that illustrates the trouble-shooting process.

Obtain and critique 3 previous peer-reviewed scientific articles/literature that describes and has pictures of novel data with protein expression and western blotting.
Present data from previously published, peer-reviewed articles.



Student Goal 2: Design a research project that is hypothesis-driven and builds upon the previous knowledge about animal-heme peroxidases, peroxidasin
 

Employ research practices to be able to perform cell culture.
Practice plotting the data.
Analyze quantifying protein bands from western blot.
Perform pippeting and practice loading of samples onto gel.



Student Goal 3: Communicate their research ideas orally and through writing

Present about peroxidases and previously identified roles of peroxidasins and point directed mutagenesis
Discuss ideas for trouble shooting and anticipated pitfalls

Practice by diagramming anticipated/expected results from western blot for protein analysis. 
Present western blot results to the class.


Instructional Materials

Cell Culture Training Videos:

Cloning Assistant Sites:

Assessment

In order to assess student learning, the instructor should speak with the student through the research process and have them maintain a lab notebook that can be reviewed often. The students should also prepare powerpoint posters for mid-semester and end of the semester presentations. These posters should be checked for title, introduction, research question, hypothesis, research methodology, results, conclusion and discussion. The students posters should be assessed (through one-on-one discussions or in-class poster or oral presentations) for learning progressions from the middle of the project to the end of the project.

Poster Design:

Here is a site to share with the students about poster design and elements from this should be used to assess their learning and poster design: http://hsp.berkeley.edu/sites/default/files/ScientificPosters.pdf

Faculty members can assess their CURES using measures described in these papers:

Instructional Staffing

1. Myself 
2. Students 
3. Faculty members 
4. Social Scientist 
5. Lab coordinator 
6. TAs 
7. Librarian 
8. Grad students
9. Collaborators or external scientists 
10. Pre-service teachers

Author Experience

Isi Ero-Tolliver, Hampton University

This cure is to assist students will learning molecular biology techniques that are transferrable across projects. These techniques will include DNA cloning, primer design, polymerase chain reaction, tissue culture, and western blot analysis.

Advice for Implementation

In order to implement this CURE, it is important that you have access to a cell culture hood, PCR machine and reagents, competent bacterial cells, waterbath and an incubator for your bacterial transformation and bacterial cell growth. Students sometimes have a hard time understanding how to design their primers and the importance of proper primer design, but by using websites such as IDT.com and the previously mentioned sites, the should be able to design proper primers that target a specific site on their DNA. To keep costs and time down, it is best to have students perform their research in groups instead of individuals and that way they can share reagents and have less freeze and thaw processes.

Iteration

Students are involved in trouble-shooting, problem-solving and repeating aspects of their work. They have the opportunity to look in the NCBI website for their DNA and protein sequences of their target construct. They also have multiple iterations at designing a primer that contains their mutant sequence and place the sequence into Netprimer software that gives them a percentage of how good their primers are (this percentage gives them a measure that is easy to read but includes Tm, GC content and much more. The students also have multiple attempts at cell culture and transfections using varying amounts of transfection reagents such as lipofectamine. If students "fail" scientifically, meaning have contamination within their cells or their mutant is not behaving properly, they have access to their instructors, online resources, and peers to discuss troubleshooting techniques and next steps. The students also use multiple cells lines for training that include HEK293 and Pfhr9 cells for different trials with different cell lines, in preparation for their DNA transfections and protein expression analysis. Trying multiple times is encouraged and necessary for learning proper techniques.

Using CURE Data

Using the data from the CURE, the instructor will be able to use results from the multiple clones to assess the crosslinking ability of peroxidasin within the basement membrane. The instructor will have results from multiple clones that either crosslink or do not crosslink the basement membrane. Therefore the instructor will be able to determine the critical amino acids within the immunoglobulin domain that is responsible for the crosslinking activity.

Resources

1.The Ancient Immunoglobulin Domains of Peroxidasin Are Required to Form Sulfilimine Cross-links in Collagen IV.Ero-Tolliver IA, Hudson BG, Bhave G. J Biol Chem. 2015 Aug 28;290(35):21741-8. doi: 10.1074/jbc.M115.673996. Epub 2015 Jul 15.

2.Peroxidasin forms sulfilimine chemical bonds using hypohalous acids in tissue genesis. Bhave G, Cummings CF, Vanacore RM, Kumagai-Cresse C, Ero-Tolliver IA, Rafi M, Kang JS, Pedchenko V, Fessler LI, Fessler JH, Hudson BG.Nat Chem Biol. 2012 Sep;8(9):784-90. doi: 10.1038/nchembio.1038. Epub 2012 Jul 29.