MAKING_LIFE 2014–2015

Artificial extremophilia
posted by sarah on 23 March 2016



Rupert Mutzel
This session involved growing wild type and experimentally evolved Escherichia coli cells at various salt concentrations.
Protocol (see below for alternative salt growth protocol)

  1. Prepare growth media (MS Mannitol) containing 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 M NaCl from stocks containing 0 and 2 M NaCl:













































Final NaCl concentration, M (approx. % w/v) Volume per 100 ml , 0 M NaCl medium Volume per 100 ml, 2 M NaCl medium
0                    (0) 100 0
0.3                 (1.8) 85 15
0.6                 (3.6) 70 30
0.9                 (5.4) 55 45
1.2                 (7.2) 40 60
1.5                 (9) 25 75
1.8                 (10.8) 10 90


  1. Label 7 tubes each for the wild type (wt) and evolved (E) strain: 0 - 0.3 - 0.6 - 0.9 - 1.2 - 1.5 - 1.8 (M NaCl)



  1. Pipet 5 ml of each medium into the corresponding tubes



  1. Inoculate each tube with a loopful of bacteria scraped from an agar plate (e.g. 3-4 agar plates per strain)



  1. Incubate at 30° C overnight with good aeration (roller or shaker)



  1. Inspect tubes for growth


 
Composition of MS (minimal salts)-Medium

































Citric acid x H20 0.84 g 4 mM
MgSO4 x 7 H2O 0.25 g 1 mM
NH4Cl 0.53 g 20 mM
K2HPO4 8.71 g 50 mM
Mannitol 2 g 11 mM
1000 x NTA-Mix 1 ml  

(H2O bidest ad 1l)
 
(1000 x N.T.A.-Mix




































































Component Concentration (M) Amount (mg)
Nitrilotriacetic acid 10-2 1910
CaCl2 x 2 H2O 3 x 10-3 450
FeCl3 x 6 H2O 2 x 10-3 810
MnCl2 x 4 H2O 10-3 200
ZnCl2 10-3 40
H3BO3 3 x 10-4 20
CrCl3 x 6 H2O 3 x 10-4 80
CoCl2 x 6 H2O 3 x 10-4 70
CuCl2 x 2 H2O 3 x 10-4 50
NiCl2 x 6 H2O 3 x 10-4 70
Na2MoO4 x 2 H2O 3 x 10-4 70
Na2SeO3 x 5 H2O 3 x 10-4 80

H2O bidest ad 1 l)
 
Alternative (salt growth) Protocol

  1. Prepare growth media (MS Mannitol) containing 0, 0.6, 1.2, 1.8 M NaCl from stocks containing 0 and 2 M NaCl:






























Final NaCl concentration (M) Volume per 100ml, 0 M NaCl medium Volume per 100 ml, 2 M NaCl medium
0 100 0
0.6 70 30
1.2 40 60
1.8 10 90

(Wild type bacteria should grow at 0 and 0.6 M salt, evolved bacteria should grow at 0.6, 1,2, 1.8 M, barely at 0 M. In advance, prepare 30 ml of each medium in sterile 50 ml Falcon tubes.)

  1. Label 4 tubes each for the wild type (wt) and evolved (E) strain: 0 - 0.6 - 1.2 - 1.8 (M NaCl)



  1. Pipet 5 ml of each medium into the corresponding tubes



  1. Inoculate each tube with a loopful of bacteria scraped from an agar plate



  1. Incubate at 30° C overnight with good aeration (roller or shaker)



  1. Inspect tubes for growth