Recombinant DNA Techniques Laboratory

Bi 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

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

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:

• 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

• 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 • 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:• 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!!!!