Recombinant DNA Techniques Laboratory Bi 431/531

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Recombinant DNA Techniques LaboratoryBi 431/531

• Introduction/Syllabus
• Class Overview
• Bioluminescent bacteria
• Micropipetting Exercise I, part I
• Aseptic techniques Exercise I, part II
• Environmental Isolates Exercise III
• Start liquid cultures for Wednesday

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Introduction/Syllabus

• Office hours
• Lab safety
• Gloves- no gloves in hallway!
• Quizzes
• Participation
• Lab notebooks
• Ink, math, up to date, random checks
• Lab reports
• Environmental isolates
• Grad presentations
• Handouts/Mol review

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Bioluminescent Bacteria

• Present in many deep sea organisms and in the
  open ocean
• Most belong to genus Photobacterium, some to
  Vibrio
• The lux operon
• 5 genes, about 8 kb
• Three genes remove Acyl ACP from fatty acid
  biosynthesis pathway
• Two genes code for the a and ß subunits of
  luciferase

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Bioluminescent Bacteria

• Quorum sensing
• Operon is turned on and off by the presence of an
  autoinducer
• acylhomoserine lactone, used by many microbes
• Expression only occurs at high cell density
  107cells/ml

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Projects

• Cloning the Lux operon
• Grow and purify DNA from cultured bioluminescent
  organisms
• Remove the lux operon with restriction
  endonucleases or PCR from genome
• Ligate into plasmid
• Transform into E.coli
• Screen for bioluminescent E. coli

• Isolation of wild bioluminescent bacteria
• Use sea samples to grow and isolate bacteria
• Create a pure stock and cryogenically freeze
• Amplify and sequence part of a lux gene to
  identify the organism

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Exercise I - Micropipetting

• Pipetter safety read and always follow
  cautionary notes on page 3
• Tubes A B
• Combine the appropriate volumes of 10X buffer,
  DNA, H2O and Enzyme ? spin ? check final volume
  (should be 10ul)
• Tubes C-F
• Combine same components along with Reagent A ?
  spin ? check final volume (should 100ul for CD,
  1000ul for EF)

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Aseptic techniques Exercise I, part II

• Streaking agar plates
• 4 different bacterial cultures will be streaked
• E.coli DH5a both LB and LB Amp plates
• E.coli DH5a (pGEM-3Zf) both LB and LB Amp
  plates
• Photobacterium leognathi PB plates
• Vibrio fisheri PB plates
• Streaking DEMO
• Broth culture inoculations
• E.coli DH5a (pUWL500 or pUWL506) LB Amp
• E.coli DH5a (pGEM-3Zf) LB Amp
• Add 40 ul of Amp to LB tubes)
• Photobacterium leognathi LBS
• Vibrio fisheri LBS

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Environmental Isolates

• Goal is to sequence the lux genes in
  environmentally isolated bacteria
• Each person will isolate their own strain
• Procedure is Ex III in Winfrey et al.
• PB plates will be used instead of LBS
• Each person will streak two PB plates from the
  provided sea creature

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Checklist

• Streaking agar plates
• E.coli DH5a both LB and LB Amp plates
• E.coli DH5a (pGEM-3Zf) both LB and LB Amp
  plates
• Photobacterium leognathi PB plates
• Vibrio fisheri PB plates
• Broth culture inoculations
• 2 E.coli DH5a (pGEM-3Zf) LB Amp Broth (for
  Wednesday Plasmid Prep)
• Photobacterium leognathi LBS
• Vibrio fisheri LBS
• 2 sea creature PB plate innoculations
• INCUBATIONS
• E.coli strains 37
• Biolumiescent strains room temperature
• FOR THURSDAY
• Read Molecular review worksheet
• Answer/hand in questions (this is this weeks
  quiz)
• Read Ex 6 Plasmid Prep (just the intro)
• Read GeneJET Plasmid Miniprep Kit instructions
  (this is what we will use)

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Recombinant DNA Techniques LaboratoryBi 431/531

• Exercise 6 Purification of Plasmid DNA with
  GeneJET Plasmid Miniprep Kit

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Large-Scale purification of Plasmid DNA

• Plasmid purification procedures take advantage of
  two differences between chromosomal and plasmid
  DNA
• Bacterial chromosomes are much larger than
  plasmids
• Unlike chromosomal DNA, plasmids are not easily
  sheared and the two strands are physically linked
  together

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purification of Plasmid DNA
The mini column
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Large-Scale purification of Plasmid DNA

• The DNA purification kit will be used
• See handout for procedure details
• Use of centrifuge
• Centrifuge safety
• Balancing
• Keeping track of pellets
• Store plasmid stocks in the appropriate box in
  the student freezer YOU WILL NEED THESE LATER!!!!