The work in the Lab – catalase out of Bacillus subtilis
The goal of our lab work was the expression of catalase in the presence of the artificial amino acids ethionine and norleucine. The laboratory bacteria used (Bacillus subtilis strain from Bacillus Genetic Stock Center, http://www.bgsc.org/) should use the artificial amino acids as part of its metabolism and subsequently produce a synthetic enzyme into the nutrient solution.
The Bacilus subtilis strain used was a methionine-, tryptophan-, tyrosine- and phenylalanine-auxotroph and thusly could not survive outside of the nutrient solution.
We wanted to take advantage of the Methionauxotrophia of the selected bacterial strain. We mixed six different nutrient solutions. Three were based on the bacterium-standard solution, so-called LB, and three based on a different standard solution called M9. We included Methonine as natural amino acid in the LBM, Norleucine in the LBN, and Ethionine in the LBE. In the same fashion, M9M, M9N and M9E were produced. LBM and M9M, each with their natural Amino acid composition, were to be our reference samples; to help us know if ours could produce amylases at all: it was the so-called Control group. The mixtures are detailed in table 1.
Even though our Bacillus strain was safe, all efforts were deliberately and carefully made to avoid contamination of the outside world. All disposable items used were inactivated in Autoclaves and Certoclaves and then thrown away. Solutions with special dyes went through a special disposal process. All organisms were also put through heat sterilization for inactivation. The lab machines, work areas, lab table etc. were disinfected with a cleaning solution and the clean box was additionally treated with UV light.
Application of the pre-culture
20 micro-liters of the dry ice stored glycerin stock of Bacilus subtilis strain 1A55 are used for the application of a pre-culture. 10 ml of the LB medium (commercially available and pre-mixed) is put into a falcon tube in the clean box. Then the medium is inoculated with the glycerin stock.
Everything must remain absolutely sterile. Every tool used is either singed, treated with UV light or cleaned with 70% ethanol, even the lab gloves.
The inoculated media are left in a vibrating incubator at 37 degrees Celsius over night. The falcon tubes should be leaned on their sides to allow for a good mixture with the air. The cap is only tight enough that it stays on. This allows for air circulation.
Determining the optical concentration
To determine the optical concentration of the probe – and the resulting amount of germs it contains – 0.9 ml of germ free nutrient medium is placed in a cuvette and then 0.1 ml of the inoculated nutrient medium is added after being thoroughly mixed. With the help of a photometer, the optical concentration at 550nm of absorption is determined. 1ml of LB medium serves as a control subject. Here, everything must also be sterile to make sure hat no foreign germs make their way into the pre-culture. The cuvette no longer needs to remain sterile. It will be thrown away.
Application of the main culture
Next, the volume of the inoculum for the main culture must be calculated. The optical concentration of the main culture should be 0.1
VInokulum = (VHK*Start OD550) / OD550VK
The nutrient solution is put into the autoclaved beaker under sterile conditions. Then the calculated inoculum is moved from the pre-culture and put into the beaker. Again, cleanly work is very important and the beaker should always be covered with aluminum foil when not in the clean box. The lip and cap of every container must also be singed with flame after every time they are opened and before every time they are closed.
The beaker with the inoculated nutrient solution is put into the
vibrating incubator at 37 degree Celsius. An agitator speed should be chosen that create a good amount of foam, thus aerating the culture.
1 ml of cell suspension should be moved to a cuvette under sterile conditions and the optical
concentration (and with it, the cellular growth) should be determined after 1, 3, 5, 7, 20, 22 and 24
hours. Optical concentration and time are important factors for the calculation of growth
curve. The samples are finally centrifuged off in the epifuge at the highest speed for 2 minutes
and the cell residue transferred with a pipette to a test tube, in which they can be stored in a cool place.
Cell Harvest and the Production of Catalase
As soon as the optical concentration begins to go down, the cells have begun to die. One must act quickly, because proteinases (=enzymes that are capable of breaking apart enzymes) can get into the solution and could destroy the desired catalase. The cell suspension is then, no longer sterile, partitioned into 50ml pipettes and put into the
centrifuge at 3,500 rpm for 10 minutes. The cellular residue is, without stirring up the cell pellet, decantered off into a filter. The cellular residue extracted in this way contains all the proteins expressed by the bacteria. The filtrate is then supplemented with ammonium sulfate to turn out the catalase. This fluid is filtered two times. With the help of a dialyzation tube, the previously added ammonium sulfate is separated from the catalase. All the proteins in the substrate are separated with an SDS gel and are cooled for later analysis with the mass spectrometer at the Max-Planck-Institute.
Table 1: Mixtures
LB medium
0.5% (w/v) yeast extract |
5 g/l |
1% (w/v) bacto tryptone |
10 g/l |
1% (w/v) NaCl |
10 g/l |
LB+ medium
0.1% (w/v) glucose |
1 g/l |
L-phenylalanin |
50 mg/l |
L-tyrosin |
50 mg/l |
L-tryptophan |
50 mg/l |
0.5% (w/v) yeast extract |
5 g/l |
1% (w/v) bacto tryptone |
10 g/l |
1% (w/v) NaCl |
10 g/l |
LBM medium
LB+ Medium |
100 ml |
L-methionin |
50 mg/l |
Dissolve methionine in LB + medium and perform sterile filtration; put 2 ml in a sterile 2 ml Eppi & store it at 4 °C; use up soon; can be stored at 4 °C overnight.
LBE medium
LB+ Medium |
100 ml |
L-ethionin |
10 g/l |
Dissolve ethionine in LB + medium and perform sterile filtration; put 2 ml in a sterile 2 ml Eppi & store it at 4 °C; use up soon; can be stored at 4 °C overnight.
LBN medium
LB+ Medium |
100 ml |
L-norleucin |
10 g/l |
Dissolve norleucine in LB + medium and perform sterile filtration; put 2 ml in a sterile 2 ml Eppi & store it at 4 °C; use up soon; can be stored at 4 °C overnight.
5x M9 concentrate
Na2HPO4·2 H2O |
42.5 g/l |
KH2PO4 |
15 g/l |
NH4Cl |
5 g/l |
NaCl |
2,5 g/l |
Adjust pH- value on 7.0 with NaOH; autoclave and store at room temperature.
100x micro elements solution
MnCl2·4 H2O |
100 mg/l |
ZnCl2 |
170 mg/l |
CuCl2·2 H2O |
43 mg/l |
CoCl2·6 H2O |
60 mg/l |
Na2MoO4·2 H2O |
60 mg/l |
Perform sterile filtration; solution should be protected from UV rays and stored at room temperature.
100 mM CaCl2
CaCl2·2 H2O |
1.47 g / 100 ml |
Autoclave and store at room temperature.
1M MgSO4
MgSO4·7H2O |
24.6 g / 100 ml |
Autoclave and store at room temperature.
50 mM FeCl3
FeCl3·6H2O |
1.35 g / 100 ml |
Autoclave and store at room temperature.
M9 medium
5x M9 concentrate |
200 ml/l |
100x micro elements solution |
10 ml/l |
100 mM CaCl2 |
1 ml/l |
1 M MgSO4 |
1 ml/l |
50 mM FeCl3·6H2O |
1 ml/l |
L-phenylalanin |
50 mg/l |
L-tyrosin |
50 mg/l |
L-tryptophan |
50 mg/l |
glucose |
5 g/l |
Fill up to 1 l with distilled water, dissolve glucose and amino acids, perform sterile filtration and use up soon, can be stored overnight at 4 °C.
M9M medium
M9 medium |
200 ml |
L-methionin |
50 mg/l |
Dissolve methionine in M9 medium and perform sterile filtration; put 2 ml in a sterile 2 ml Eppi & store it at 4 °C; use up soon; can be stored at 4 °C overnight.
M9E medium
M9 medium |
200 ml |
L-ethionin |
50 mg/l |
Dissolve ethionine in M9 medium and perform sterile filtration; put 2 ml in a sterile 2 ml Eppi & store it at 4 °C; use up soon; can be stored at 4 °C overnight.
M9N medium
M9 medium |
200 ml |
L-norleucin |
50 mg/l |
Dissolve norleucine in M9 medium and perform sterile filtration; put 2 ml in a sterile 2 ml Eppi & store it at 4 °C; use up soon; can be stored at 4 °C overnight.