Recombinant DNA Techniques Laboratory Bi 431/531

1. 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

2. 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

3. 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

4. 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

5. 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 Projects

6. 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)

7. 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

8. 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

9. 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)

10. Recombinant DNA Techniques LaboratoryBi 431/531 • Exercise 6 –Purification of Plasmid DNA with GeneJET™ Plasmid Miniprep Kit

11. 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

12. purification of Plasmid DNA The mini column

13. 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!!!!