1. 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

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. 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

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 Broth culture inoculations
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 Laboratory Bi 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!!!!