MAKING_LIFE 2014 - 2015

Artificial extremophilia
posted by sarah on 23 March 2016

W_2_day2 (33)

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)

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




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)


Pipet 5 ml of each medium into the corresponding tubesW_2_day2 (1)


Inoculate each tube with a loopful of bacteria scraped from an agar plate (e.g. 3-4 agar plates per strain)W_2_day2 (35)


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


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

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.)

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


Pipet 5 ml of each medium into the corresponding tubes


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


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


Inspect tubes for growth