Team:Cambridge/Bacillus/Lab Work

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July 22nd

We have 4 tubes from last year. These strains are frozen in glycerol.

  • PNZ8901 plasmid in E.coli MC1061 Strain
    • Chloroamphenicol resistant
    • "sure" vector
    • does not integrate
  • B. subtilis strain 1A1
    • No resistance
    • Same as strain 168 but tryptophan deficient
  • E.coli strain MC1061, no plasmid
  • PP182 plasmid in E.coli strain DH5alpha
    • Ampicillin resistance
    • integrates


Checking antibiotic resistance

Purpose : Grow each of them on a plate to test their antibiotic resistance

Protocol

  • Warm up frozen tubes
  • Take 15uL of each and put it on plates without antibiotic
  • Incubate at 37°C


July 24th

  • Test of antibiotic resistances of strains of last year
Strain Plasmid Extracted from Antibiotic use Observations Conclusion
DH5alpha PP182 tube Amp Many colonies Amp Resistance OK
DH5alpha PP182 plate Amp Many colonies Idem
MC1061 NONE tube Cm Nothing As expecting, no Cm Resist.
MC1061 NONE plate Cm Nothing Idem
MC1061 PNZ8901 plate Cm Maybe a few colonies Contamination??
MC1061 PNZ8901 tube Cm No colonies no Cm Resist.


  • Transformation of E.coli with different plasmids from last year
Strain Inserted plasmid Antibiotic Observation Conclusion
E.coli I746000 Amp No colonies Antibiotic resistance unknown, no Amp Resist., Pb : no terminator
E.coli I746100 Amp No colonies Antibiotic resistance unknown, no Amp Resist., Pb : no terminator
E.coli I746001 Amp Many colonies Transformation OK
E.coli I746101 Amp Many colonies Transformation OK

Antibiotic test for Bacillus resistance

We want to find the lowest concentration of antibiotic which kills Bacillus.


Dilution of Amp. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]

Concentration (μg/mL) 100 75 50 25 10
100 mg/mL Stock 1:1000 3:4000 1:2000 1:4000 1:10000

Dilution of Cm. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]

Concentration (μg/mL) 35 25 15 10 5
100 mg/mL Stock 1:1000 1:1400 3:7000 1:3500 1:7000
  • Add Disks in antibiotic solution for 5 mins [ Protocol to sterilize tweezers : Wipe with Kimwipes, Ethanol, Flame ]
  • Melt Soft Agar
  • 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08
  • Pour SA+Cells over blank hard agar plates
  • put disks with different concentration of Amp/Cm above SA
  • Incubate at 37°C

Results [Plates were prepared before lunch, at 5pm there were visible growth]

-Amp Plates: Huge rings of no growth around 100, 75, 50, 25, and 10.

-Cm PLates: Tiny rings of no growth around 5, 10, 15 and 25. Small ring of no growth around 35 but not well defined. (No clear zones)


Salts for making Bacillus Competent

10x Medium A Base

  • Yeast Extract 10g
  • Casamino Acid 2g
  • add Distilled water to 900mL

-Aliquot into 5 different bottles. [180mL each]

10x Bacillus Salts

  • NH4)2SO4 20g
  • K2PO4 anhydrous 139.66g
  • KH2PO4 60g
  • Tri-Sodium Citrate 10g
  • MgSO4.7H2O 2g
  • Add Distilled water to 1000mL

-Aliguot into 5 different bottles. [200mL each]

Medium B

    • Preparing 50mM CaCl2.2H2O
      • CaCl2.2H2O 1.470g
      • Add Distilled water to 20mL [Final conc. 500mM]
      • Take 10mL of 500mM
      • Add 90mL of distilled water [Final conc. 50mM]
    • Preparing 250nM MgCl2.6H2O
      • MgCl2.6H2O 0.508g
      • Add Distilled water to 10mL [Final conc. 250mM]
      • Take 1mL of 250mM
      • Add 99mL of Distilled water [Final conc. 250μM]
      • Take 1mL of 250μM
      • Add 99mL of Distilled water [Final conc. 250nM]
  • Add 100mL of 50mM CaCl2.2H2O
  • Add 100mL of 250nM MgCl2.6H2O

-Total volume 200mL

NOTE: To complete Medium B, Take 0.2mL of this solution

July 23rd

  • Take one colony from each of the plates grown from the tubes yesterday and plated them again on LA without antibiotics


1/ Purpose Check antibiotic resistance of our different strains

Strain Plasmid Extracted from Antibiotic use Concentration of antibiotic
DH5alpha PP182 tube Amp 100
MC1061 NONE tube Cm 35
MC1061 PNZ8901 tube Cm 35
DH5alpha PP182 plate Amp 100
MC1061 NONE plate Cm 35
MC1061 PNZ8901 plate Cm 35


2/ Purpose : Find the right concentration of antibiotic so that B. subtilis survive

- Grow 15uL of B. 1A1 (frozen tube) in 5mL of LB

- Incubate at 37°C

3/ Purpose : Grow plasmids in TOP10, transformation

4 plasmids :

  • I746000
  • I746100
  • I746101
  • I746001

1 control : PUC19

- Add 20uL of TOP10 competent cells and 0.5uL of Plasmid in an eppendorf

- 30min on ice

- Heat shock : 60s at 42°C

- 2min on ice

- Add 89.5uL of SOC

- 60min at 37°C

- Put the mix on plate with ampicillin resistance

- Incubate at 37°C





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