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pGLO Transformation

MCB 151 FA19

zAnnouncements

§ Exam 2 will take place in LON-CAPA on April 20.

§ You will have one hour to complete it

§ Your exam clock begins when you open the first question

§ Final Exam is Friday, May 8. Also in LON-CAPA.

§ Conflict Final Exam, must fill out the online form.

zGenetic Engineering

§ The manipulation of an organism’s genetic material (DNA) by introducing or eliminating specific genes

zGFP

(Green Fluorescent Protein)

• Originates from the bioluminescent jellyfish

• Allows the jellyfish to fluoresce and glow-in-the-dark

• Following successful transformation your bacteria will express their

newly acquired jellyfish gene and produce the fluorescent protein

causing them to glow under UV light

zPlasmid DNA

(pGLO Plasmid)

• Beta Lactamase (bla) contains the ampicillin resistance gene

• GFP from Aequorea victoria(jellyfish)

• araC is a regulator protein. Regulating GFP transcription. If arabinose is present, then GFP is transcribed

zTransformation of E. coli

1. Move the pGlo plasmid DNA through the cell membrane using CaCl2

2. Ice treatment

3. Heat shock @ 42°C

4. Ice treatment

zPlate Labeling

zSpread Plating Transformed Cells

zExpected Results

zTransformation Efficiency

Total number of cells growing on the agar plate

Amount of DNA spread on the agar plate

zObservations and Completion of LNAs

§ Students will receive an email with their results at the end of the week. Observations and colony counts will need to be completed from these photos.

§ LNAs are due at the START of your next lab period (week of November 11).

zAssignments and Looking Ahead

§ LNA pGlo due at the start of lab, week 11 (April 12)

§ Exam 2 (April 20)

§ Final Exam – May 8

§ Gradebook Deadline – May 6 @ 5 PM

§ Conflict Final Exam Request Deadline – May 6, 5 PM