Griffitts:MMBIO 460

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Contents

The Class

The purpose of this class is to train students in the basic techniques of bacterial genetics. It consists of lectures on Mondays and Fridays and a three-hour lab on Wednesdays with a follow-up lab on Thursdays. The first half of the semester has a structured lab set-up to provide the students with the bacterial geneticist's toolbox. During the second half, the students are expected to take what they've learned and construct their own project, complete it, and present their data to the class. The structured lab activities are as follows:

  1. Basic Skills
  2. Mutagenesis
  3. Conjugation
  4. Transposition
  5. Recombination (Part I)
  6. Recombination (Part II)
  7. Screen for Regulatory Mutants

Set-up

The following is a description of the set-up of each lab along with some miscellaneous notes and observations. The following are generally needed for every lab and should be set out beforehand:

  • Pipetmen (P20, P200, P1000)
  • Pipetman tips (sterile)
  • microcentrifuge tubes (sterile)
  • toothpicks (sterile)
  • waste containers
  • ethanol
  • Bunsen burners (1 per 2 students)
  • lighters
  • spreaders (one per student)
  • ice buckets
  • LB broth

Basic Skills

Materials needed:

  • An overnight culture of B079 in 25 mL of plain LB
  • The following bacterial strains on agar plates (preferably plated out fresh the night before):
    • B079 on plain LB
    • B081 on plain LB
    • B381 on plain LB
    • B392 on plain LB
  • The following agar plates:
    • LB (6 per student)
    • LB-Sm (1 per student)
    • SMM-Glc (1 per student)
    • SMM-Lac (1 per student)

Procedure

  • Students perform a dilution series of the overnight B079 culture and plate out the 10-5, 10-6, and 10-7 dilutions.
  • Students streak the B079 to singles on LB agar
  • Students streak B079, B081, B381, and B392 on LB and LB-Sm plates to determine Streptomycin susceptibility
  • Students streak B079, B081, B381, and B392 on SMM-Glc and SMM-Lac plates to determine lactose utilization capabilities.

Mutagenesis

Materials needed (Wednesday):

  • 2-day passaged culture of B079 in 25 mL plain LB
    • Use a large flask to ensure aeration
    • Shake at 250-300 rpm
    • Passage 1: 10 μL into 25 mL fresh LB
    • Passage 2: 10 μL into 50 mL fresh LB
  • Sodium nitrite (0.4 M)
  • Sodium acetate buffer (0.1 M, pH 4.6)
  • The following agar plates:
    • LB (8 per student)
    • LB-Sm (4 per student)
  • LB broth + 10% glycerol

Procedure (Wednesday):

  • Students perform mutagenesis with 4 tubes of 500 μL of the B079 culture
    • Centrifuge for 30 seconds at full speed
    • For experiment, resuspend with 450 μL sodium acetate + 50 μL sodium nitrite in two tubes
    • For control, resuspend with 500 μL sodium acetate in two tubes
    • Let one experimental tube and one control tube go 15 minutes and the others 20 minutes
    • Centrifuge, resuspend in LB, and allow 1.5 hours recovery time
    • Plate appropriate dilutions from all four tubes
    • Centrifuge, resupend in LB + 10% glycerol, and freeze at -80°C
  • Students evaluate the spontaneous appearance of streptomycin resistance
    • Centrifuge 4 tubes with 1.5 mL of the B079 culture
    • Resuspend in 100 μL LB and spread on LB-Sm plates

Materials needed (Thursday):

  • The following agar plates:
    • LB-XGal (11 per student)
    • LB-XGal-IPTG (1 per student)
  • The following bacterial strains on agar plates (preferably plated out fresh the night before):
    • B079
    • B382
    • B383

Procedure (Thursday)

  • Students calculate viable CFUs in their mutagenized B079
  • Students perform the proper dilution series and plate 2000 CFUs/plate onto to plates of LB-XGal
  • Students plate B079, B382, and B383 on LB-XGal for "blue" control
  • Students plate B079, B382, and B383 on LB-XGal-IPTG for "white" control

Conjugation

Materials needed (Wednesday)

  • The following bacterial strains on agar plates (must be plated out fresh the night before):
    • B001 on LB-Cm
    • B100 on LB-Sm (plate this one out 2 days in advance as it grows more slowly)
    • B393 on LB-Sm
    • pJG100 on LB-Km
    • pJG194 on LB-Km
    • pJG211 on LB-Km
    • pJG247 on LB-Km
  • The following agar plates:
    • LB (4 per student)

Procedure (Wednesday)

  • Students perform triparental matings and control matings on LB agar
    • B001 is the helper strain
    • B100 (S. meliloti) and B393 (E. coli) are the recipients
    • pJG100, pJG194, pJG211, and pJG247 are the donors
    • Controls are performed by excluding the helper strain (B001)

Materials needed (Thursday)

  • The following agar plates:
    • LB-Sm-Km (2 per student)
    • LB-Sm-Nm (2 per student)

Procedure (Thursday)

  • Students plate matings with B100 onto LB-Sm-Nm plates
  • Students plate matings with B393 onto LB-Sm-Km plates

Transposition

Materials Needed (Wednesday)

  • The following bacterial strains:
    • B001 on LB-Cm
    • B100 on LB-Sm
    • pJG110 on LB-Ap
    • pJG210 on LB-Ap
  • The following plates:
    • LB (2 per student)

Procedure (Wednesday)

  • Students perform triparental matings and control matings in LB broth
    • B001 is the helper strain
    • B100 (S. meliloti) is the recipient of pJG110 (donor) and pJG210 (donor)
    • Prepare 100 μL of the four strains by transferring globs into LB broth with toothpicks in Eppendorf tubes
    • Prepare two tubes of B100
    • Combine 10 μL of B001 and 10 μL of pJG110 to one tube of B100
    • Combine 10 μL of B001 and 10 μL of pJB210 to the other tube of B100
    • Plate 100 μL of each mating onto LB plates

Materials Needed (Thursday)

Procedure (Thursday)

  • Students plate matings onto LB-Sm-Nm-XGal-Succinate plates (10, 1.0, and 0.1 μL)

Recombination (Part I)

Materials Needed for Electroporation (Wednesday)

  • Electrocompetent B451 cells (50 μL per student) for λ-red knockouts
    • This should be done the morning of
    • Grow B451 at 30°C overnight in 25 mL LB-Ap broth
    • Inocculate 100 mL LB-Ap broth with 250 μL of the overnight culture
    • Shake at 30°C until OD600 = 0.2
    • Add 1 mL of 1M arabinose (or 750 μL of 20% arabinose)
    • Continue shaking at 30°C until OD600 = 0.5
      • After this point, keep all cultures and liquids on ice
    • Spin at 8000 rpm for 10 minutes in cold SS34 rotor
      • The Sorvall Centrifuge is found in the Harker/Breakwell lab (747 WIDB)
      • Divide the culture into 2 tubes with ~40 mL each
    • Remove all supernatant
    • Gently resuspend with 15 mL cold 10% glycerol
    • Spin at 8000 rpm for 10 minutes
    • Remove all supernatant
    • Gently resuspend with 15 mL cold 10% glycerol
    • Spin at 10,000 rpm for 10 minutes (higher rpm?)
    • Remove all supernatant (the pellet is especially fragile at this point)
    • Gently resuspend with ~200 μL cold 10% glycerol and combine
      • Time this so that you can immediately place the cells on ice and the students can begin to set up the electroporation
  • Linear DNA solution
  • The following plates:
    • LB-Km (1 per student)

Materials Needed for Phage Transformation (Wednesday)

  • P1 phage
  • LB-Top Agar + CaCl3 (1 75-mL bottle per 5 students)
  • 15-mL centrifuge tubes (1 per student)
  • The following bacterial strains
    • B402 (Aux 1) on LB-Km
    • B403 (Aux 2) on LB-Km
    • B404 (Aux 3) on LB-Km
    • B405 (Aux 4) on LB-Km
    • B406 (Aux 5) on LB-Km
    • B479 (MG1655 + pJG100) on LB-Km (as control)
    • B480 (fliC-) on LB-Km (as back-up)
    • B481 (fliC-) on LB-Km (as back-up)
    • B482 (fliC-) on LB-Km (as back-up)

Procedure for Electroporation (Wednesday)

  • Students electroporate DNA into competent B451 cells
    • Add 1.5 μL linear DNA solution to 50 μL electrocompetent cells
    • Transfer electrocompetent cells with added DNA into a chilled electroporation cuvette
    • Shock
      • The electroporator is in Dr. Erickson's lab (??? WIDB)
      • Use the EC1 setting
    • Immediately add 400 μL LB broth and pipet up and down
    • Transfer to an Eppendorf tube
    • Place on shaker at 30°C for 1 hour
    • Plate 10 μL onto LB-Km plates

Procedure for Phage Transformation (Wednesday)

Materials Needed (Thursday)

  • The following plates:
    • LB-Swarm Agar (2 per student)
      • Note: Try a higher amount of agar. This time the control was hazy and hard to distinguish; with more agar, it might be tighter and easier to see.
  • The following bacterial strains:
    • B451 on LB-Ap

Procedure for Electroporation (Thursday)

  • Students stab recovered colonies into LB-Swarm Agar
  • Students stab B451 (control) into LB Swarm Agar
  • Incubate at 30°C

Procedure for Phage Transformation (Thursday)

  • Students harvest phage particles
    • Add 2 mL LB broth onto LB-Top Agar
    • Crush agar using a spreader bar
    • Remove 1 mL and transfer to an Eppendorf tube
    • Centrifuge at 14,000 rpm for ~30 seconds
    • Remove 800 μL supernatant and add to a new Eppendorf tube
    • Add 50 μL chloroform
    • Store at 4°C

Recombination (Part II)

Materials Needed (Wednesday)

  • The following agar plates:
    • LB-Km (2 per student)
  • An active culture of B079
    • Start an overnight culture on Tuesday
    • Inncolate 100 μL of culture into 40 mL LB broth at 10 AM Wednesday morning
    • Place on a shaker at 250 rpm at 37°C
  • P1 lysates from the previous week

Procedure (Wednesday) Materials Needed (Thursday)

  • The following bacterial strains:
    • B079 on LB
    • B081 on LB
    • B402 on LB-Km
    • B403 on LB-Km
    • B404 on LB-Km
    • B405 on LB-Km
    • B406 on LB-Km
  • The following agar plates
    • LB (1 per student)
    • LB-Sm (1 per student)
    • SMM-Glucose (1 per student)

Procedure (Thursday)

  • Students pick colonies from their plates and transfer them to LB, LB-Sm, and SMM-Glucose plates

Screen for Regulatory Mutants

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