Transcript Recombinant DNA Techniques Laboratory Bi 431/531

Recombinant DNA Techniques
Laboratory
Bi 431/531
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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
Introduction/Syllabus
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Office hours
Lab safety
Gloves- no gloves in hallway!
Quizzes
Participation
Lab notebooks
– Ink, math, up to date, random checks
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Lab reports
Environmental isolates
Grad presentations
Handouts/Mol review
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 α and ß subunits of luciferase
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
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
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 C&D, 1000ul for E&F)
Aseptic techniques – Exercise I,
part II
• Streaking agar plates
– 4 different bacterial cultures will be streaked:
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E.coli DH5α – both LB and LB Amp plates
E.coli DH5α (pGEM-3Zf[+]) – both LB and LB Amp plates
Photobacterium leognathi – PB plates
Vibrio fisheri – PB plates
– Streaking DEMO
• Broth culture inoculations
• E.coli DH5α (pUWL500 or pUWL506)– LB Amp
• E.coli DH5α (pGEM-3Zf[+]) – LB Amp
– Add 40 ul of Amp to LB tubes)
• Photobacterium leognathi – LBS
• Vibrio fisheri – LBS
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
Checklist
• Streaking agar plates
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E.coli DH5α – both LB and LB Amp plates
E.coli DH5α (pGEM-3Zf[+]) – both LB and LB Amp plates
Photobacterium leognathi – PB plates
Vibrio fisheri – PB plates
• Broth culture inoculations
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2 E.coli DH5α (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:
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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)
Recombinant DNA Techniques
Laboratory
Bi 431/531
• Exercise 6 –Purification of Plasmid DNA
with GeneJET™ Plasmid Miniprep Kit
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
purification of Plasmid DNA
The mini column
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!!!!