Genetic Engineering and Cloning
  • An introduction to genetic engineering and protein expression
MINT Learning Objectives

  • Explain what a restriction site is

  • Distinguish between sticky and blunt ends following restriction digestion, and explain why sticky ends promote cloning

  • Outline what is happening in a PCR reaction (Denaturing, annealing, extension)

  • Describe the purpose of primers in a PCR reaction

  • Correlate the amplification of a target sequence via PCR to the ability to clone one gene from a much larger genome

  • Explain the role of plasmids in a cloning scheme, by describing the features of plasmids and their purposes

  • Describe how primers that include engineered restriction sites enable cloning

  • Outline the processes needed in order to clone a target gene

National Standards Alignments:

  • Vision and Change Core Concepts and Competencies (http://visionandchange.org)

    • Core Concept:

      • Information Flow, Exchange, and Storage: The growth and behavior of organisms are activated through the expression of genetic information in context

    • Core Competencies:

      • Ability to apply process of science: Biology is evidence based and grounded in the formal practices of observation, experimentation, and hypothesis testing.

      • Ability to use modeling and simulation: Biology focuses on the study of complex systems

  • Biocore Guide (Brownell et al., https://doi.org/10.1187/cbe.13-12-0233)

    • Information Flow: In most cases, genetic information flows from DNA to mRNA to protein, but there are important exceptions.

    • Information Flow: Individuals transmit genetic information to their offspring; some alleles confer higher fitness than others in a particular environment.

  • Process of Science Skills, Pelaez, N, et al. “The Basic Competencies of Biological Experimentation: Concept-Skill Statements“ (2017). PIBERG Instructional Innovation Materials. Paper 4. http://docs.lib.purdue.edu/pibergiim/4

    • Representations

      • Diagram/cartoon the steps of an experimental method 

      • Construct a visual representation (e.g. a graph or diagram) of predicted results

Interactive Video Vignette Information
IVV Title: Send in the Clones (Draft Form)
IVV URL for Students:  
  • Copy the URL to the Clipboard. Either paste it into an email to your students, or use it to create a link in your course management system.
IVV Description
While preparing for their lab, two students model the steps of a molecular cloning experiment. This introduces the viewer to restriction digestion, sticky and blunt ends, PCR, primer design, plasmids, multi cloning sites, and antibiotic selection. Viewer understanding is promoted via interactive questions embedded throughout the IVV.
Novice Ideas and IVV Learning Goals
Novice Ideas

  • Students conflate cloning of organisms with cloning of genes.

  • Students do not know the steps/processes involved in cloning genes.

  • Students lack an appreciation of scale and thus are unaware of the numbers of molecules involved in the process.

Ideas addressed in the IVV

  • PCR and well-designed primers allows you to amplify a single gene from a complex genome.

  • To amplify a gene means to make billions of copies of that gene.

  • Restriction enzymes are enzymes which can recognize a specific DNA sequence, and will cut the DNA backbone of both strands in the double helix to produce sticky or blunt ends.

  • Sticky ends with complementary base sequences are used to align and hold two pieces of DNA together in order for ligase to seal the DNA backbone

  • Plasmids are naturally occurring circular genetic elements that bacteria will use to share genes with other bacteria.

  • Cloning means to make many copies of the original thing (cell, gene, etc.)

  • Scientists have added and deleted sections of naturally occurring plasmids to make plasmids that can be used to clone genes.

  • Primers can be designed and made with a restriction site at the 5’ end. This lets researchers to add a restriction sites on the ends of the final PCR product. If the two ends have different enzyme sites, then you can clone a gene into your plasmid in the right direction to be able to express it.

  • E. coli is a bacterium that is commonly used in cloning.

  • E. coli doubles rapidly, making it an ideal ‘factory’ to produce very large numbers of our plasmids.

  • Engineered plasmids need to have a selectable marker, such as an antibiotic resistance gene.

Recommended In-class Curricular Material
Please see the MINT FAQ page for general information on the use of MINTs and IVVs with your class. The following curricular materials are provided as examples of resources that may be used in class to further student learning towards IVV and MINT learning objectives.
Assessment Question Information
The research team has developed multiple select questions for assessing IVV effectiveness. Please contact the research team if you are interested in assessing IVV use in your course.
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Acknowledgement

This material is based in part upon work supported by National Science Foundation (NSF) grants 1432286 and 1432303. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

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