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Molecular Cloning

Dive into recombinant DNA technology with cell division, transcription and translation. Includes concepts in restriction enzymes, cloning and reporter genes.

About This Simulation

Molecular cloning is one of the techniques that has laid the foundation for modern biotechnology. The technique was first used in the 1980′s and allowed the insertion of an insulin gene derived from humans to be inserted into yeast and coli bacteria. This allowed the microbes to produce insulin, which is the primary medication in diabetes treatment. Since then, molecular cloning and genetic engineering has become one of the most fundamental techniques ranging from pharmaceutical production, bioethanol production along with medical to basic research.

In this lab, the student learns how to assemble an expression vector containing TetOff regulator, RAD52 and GFP. The aim is to control the expression level of RAD52 with Doxycyline and to monitor the expression level by observing the GFP signal.

Vector assembly

In this module, students will learn how to extract DNA from yeast cells and restrict enzyme isolation in DNA from another vector. Students will prepare the extracted DNA and measure the concentration. After this, students will assemble a vector containing a TetOff regulator, gene of interest (RAD52) and GFP using the correct ligase, buffer and temperature of incubation.

Transformation

The assembled vector will be transformed into yeast cells using electroporation. RAD52 gene expression is regulated using the TetOff regulator. When Doxycyline is added to the media, RAD52 gene will be silenced. GFP is used as a reporter gene to RAD52, cells with active RAD52 will also express GFP and cells with silenced RAD52 will not express GFP. The GFP signal is monitored by exposing the cells to blue light.

DNA damage and repair system

RAD52 is hypothesized to be an important player in DNA repair. Students will perform an experiment comparing the result of induced DNA damage through UV radiation in cells expressing RAD52 and cells with silenced RAD52. If RAD52 is important in performing DNA repair, cells with silenced RAD52 will not survive the UV radiation treatment.

By going through the Molecular Cloning lab, students will get an overview of the molecular cloning techniques and the reporter gene. Moreover students will also learn about DNA damage and DNA repair system.

Screenshots

Collaborators

Rasmus Frandsen, Bioengineering Associate Professor

Rasmus Frandsen, Bioengineering Associate Professor

Technical University of Denmark (DTU)

Learning Objectives

  • Understanding molecular cloning techniques: DNA extraction and preparation, ligation, transformation, plate streaking and antibiotic selection
  • Understanding inducible gene expression regulation (TetOff system)
  • Understanding the use of GFP as a reporter gene
  • Understanding DNA damage and DNA repair system

Techniques In This Simulation

  • Tet-off gene
  • DNA extraction
  • Transformation
  • Colony screening
  • Cloning

Collaborator

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