Protocols

Recipes for Solutions and Media

2x YT medium

16 g	Bacto tryptone
10 g	Bacto yeast extract
5 g	NaCl

were dissolved in 1 L dH₂O, the pH was adjusted to 7.0.
For solid medium 12 g of Bacto agar (per Liter) was added.

LB medium

10 g	Bacto tryptone
5 g	Bacto yeast extract
10 g	NaCl

were dissolved in 1 L dH₂O, the pH was adjusted to 7.0.
For solid medium 12 g of Bacto agar (per Liter) was added.

NMS medium

1 g	MgSO4 · 7 H2O
0.2 g	CaCl2 · 6 H2O
2 mL	Chelated Iron Solution
1 g	KNO3
0.5 mL	Trace Element Solution
0.272 g	KH2PO4
0.717 g	Na2HPO4 · 12 H2O
12.5 g	Purified Agar
1 L	Distilled deionized water

Adjust pH to 6.8.

PBS

0,8 % (w/v)	NaCl
0,02 % (w/v)	KCl
0,144 % (w/v)	Na2HPO4 · 2 H2O
0,024 % (w/v)	KH2HPO4

Chelated Iron Solution

0.1 g	Ferric (III) ammonium citrate
0.2 g	EDTA, sodium salt
0.3 mL	HCl (concentrated)
100 mL	distilled deionizied water

Trace Element Solution

500 mg	EDTA
200 mg	FeSO4 · H2O
10 mg	ZnSO4 · 7 H2O
3 mg	MnCl2 · 4 H2O
30 mg	H3BO3
20 mg	CoCl2· 6 H2O
1 mg	CoCl2· 6 H2O
2 mg	NiCl2 · 6 H2O
3 mg	Na2MoO4 · 2 H2O
1 L	Distilled water

50x TAE Buffer stock solution

242 g	2 M Tris base
51,1 mL	glacial acetic acid (17,4 M)
200 mL	100 mM EDTA (pH 8)

were dissolved in 1 L dH₂O.

Loading buffer for gel electrophoresis
A 0.2 M EDTA and 0.05 % (w/v) orange G or bromphenoleblue solution was prepared in 50 % glycerine.

Ampicillin stock solution
10 g ampicillin sodium salt was dissolved in 100 mL dH₂O, sterile filtered, aliquoted and stored at -20 °C.
 

Kanamycin stock solution

5 g kanamycin sulfate was dissolved in 100 mL dH₂O, sterile filtered, aliquoted and stored at -20 °C.

Chloramphenicol stock solution
3.4 g chloramphenicol was dissolved in 100 mL Ethanol, sterile filtered, aliquoted and stored at -20 °C.

Tetracyclin stock solution
5 g Tetracyclin was dissolved in 100 mL Ethanol, sterile filtered aliqoted and stored at -20 °C.
 

TFB 1, pH 5.7 for preparation of competent cells

0.3 g	CaCl2
0.6 g	potassiumacetate
1.2 g	RbCl
1 g	MnCl2 · 4H2O
15 ml	glycerin

were dissolved in dH₂O and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.

TFB 2, pH 8.0 for preparation of competent cells

2.2 g	CaCl2
0.12 g	potassiumacetate
0.21 g	morphoslinopropanesulfuricacid
15 ml	glycerin

were dissolved in dH₂O and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.
 

5x Laemmli Buffer

10 % (w/v)	SDS
50 % (w/v)	Glycerine
0.02 % (w/v)	Bromphenol blue
15 % (w/v)	Beta-Mercaptoethanol

Acrylamide - Mix

30 % (w/v)	Acrylamide
0.8 (w/v)	Bisacrylamide

 

List of used strains

Strain	Genotype	Risk Group	Risk to Humans
E. coli XL1-Blue-MRF‘	endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+ lacIq Δ(lacZ)M15 Amy CmR] hsdR17(rK- mK+)	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys
Methylococcus capsulatus	wildtype	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys
E. coli BL21 DE3	F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys
E. coli JM109	endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36 proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)	S1	Non-pathogenic, may cause irritation to skin, eyes and respiratory tract, may affect kidneys

List of used Primers

The primer sequences that were used.

E. coli Culture Conditions

For the cultivation of E. coli 2xYT medium was used. If necessary 1 µL/mL of an antibiotic stock solution was added. The cultures were incubated at 37 °C and in case of liquid cultures shaken at 250 rpm.

E. coli Continous Cultivation

Continuous cultivations were conducted in a 2 L stirred tank bioreactor system (Applikon) with one six-bladed disc turbine impeller. Bioreactors were inoculated with mixed cultures which were derived from mixing different ratios of overnight monocultures to an OD₅₂₀ of 0.5. Growth temperature (37 ± 0.1°C), aeration rate (2.5 L  min⁻¹), agitation speed (350 min⁻¹) and pH value (pH 7.0) were automatically kept constant. The working volume was 1 L and the dilution rate was adjusted to 0.5 h⁻¹.
Depending on the mode of growth (regulated or unregulated) the medium was supplemented with ampicillin or chloramphenicol, respectively. Samples were taken every hour to monitor the cell density. To determine the ratios of the two strains in the culture, samples were spread out on agar plates, incubated at 37 °C and obtained colored colonies were counted.

Methylococcus capsulatus Culture Conditions

For the cultivation of M. capsulatus NMS medium was used. The cultures were incubated at 42 °C at 50 % methane atmosphere.

Measurement of Growth Curves

Overnight cultures in 2xYT medium containing the appropriate antibiotic were inoculated directly from -80 °C glycerol cell stock. Cells were grown in non-baffled-flasks at 37 °C and 250 rpm. The next day, main cultures in 2xYT medium containing the appropriate antibiotic were inoculated from overnight cultures to an OD₆₀₀ 0.1. Cells were grown in non-baffled flasks at 37°C and 250 rpm. Samples from each flask were taken at appropriate time points depending on the growth phase of the cells. Optical density was measured at a wavelength of 600 nm. Cells were left to grow until the stationary phase was reached.

Preparation of chemically competent cells

100 mL of 2xYT media containing required antibiotic were inoculated and incubate at 37 °C and 250 rpm until OD₆₀₀ 0.5. Subsequently the cells were incubated on ice for 15 minutes and then centrifuged at 4 °C and 400 rpm for 5 minutes. The excess was discarded and the pellets resuspended in 7.5 mL ice cold TFB1. Afterwards the solution is incubated on ice for 90 minutes and then centrifuged for 5 minutes at 4000 rpm. Finally the pellets were resuspended, aliquoted and quick-frozen in liquid nitrogen. The vials were stored at -80°C.

Cryopreservation

The respective strain was grown on 2xYT medium at 37 °C and 250 rpm overnight. Afterwards a 20 % Glycerin solution was prepared with the liquid culture, aliquoted, shock frozen and stored at -80 °C.

Minipreparation of Plasmid DNA

Miniprep was done with Plasmid DNA purification Kit by Macherey-Nagel following the manufacturer’s protocol. First 3-5 mL of the cell suspension were spun down for 10 minutes (5000 g). For the alkaline lysis of the DNA the pellet was resuspended in 250 μL Resuspension Buffer A1. 250 μL of Lysis Buffer A2 was added and incubated at RT for 5 min. 300 μL of Neutralization Buffer A3 was added to stop the reaction. As final step of the alkaline lysis the mixtures was centrifuged at 10.000 x g for 10 minutes. Afterwards the NucleoSpin Column was loaded with 750 μL lysate and then centrifuged for 1 minute (10.000 x g). Subsequently the DNA was washed with 500 μL Wash Buffer and centrifuged again for 1 minute (10.000 x g). The DNA was the precipitated with the Ethanol containing wash buffer and separated by another centrifugation (1 min, 10000 x g) and dried. At last the DNA was eluted with 50 μL elution buffer, which was pre-heated to 60 °C, and incubated for 5 min. The eluted DNA was separated from the matrix by a final centrifugation step (1 min, 5000 x g).

Colony PCR

After successful transformation single colonies were picked and analyzed by colony PCR. Therefore each colony was picked from the original plate with a pipette tip and dipped into the PCR-mix. Afterwards the pipette tip was used to inoculate a second agarplate. PCR fragments were analyzed via gelectrophoresis.

Component	μl per 10 μL reaction batch
GoTaq Buffer flexi green	2
dNTP mix	0.2
MgCl2 (25 mM)	0.8
Primer 1	0.5
Primer 2	0.5
Promega GoTaq polymerase	0.05
H2O	5.95

 

Temperature [°C]	Time	Cycles
95	2 min	1
94	15 s	30
52	20 s
72	60 s
72	10 min	1

DNA amplification via PCR with NEB Phusion High-Fidelity DNA Polymerase

Component	μl per 20 μl reaction batch
Nuclease- free water	add 20
5x Phusion HF	4
10 mM dNTPs	0.4
10 μM forward primer	1
10 μM reverse primer	1
Template DNA	Variable: 1 pg - 10 ng
Phusion DNA polymerase	0.2

 

Temperatur [°C]	Time	Cycles
98	30 s	1
98	10 s	30
50 - 60 depending on primer	30 s
72	30 s/kb
72	10 min	1

 

Gel electrophoresis

Depending on the size of the DNA fragments a 0.8 to 2 % agarose gel with 0.2 µg/mL ethidiumbromide was used. The gel was placed in an electrophoresis chamber and covered in 1x TAE buffer. Subsequently DNA samples mixed with loading buffer were loaded to the gel pockets and separated at 80-120 V for about 1 h. Last the gel was photographed for documentation.

Gel Slice Preparation

10 µL of 6x loading buffer were added to digested DNA und mixed. The sample was loaded onto a 1 % agarose gel and separated via gel electrophoresis at 75 mA for 20-40 min depending on the size of the DNA. For gel extraction the gel was visualized on an UV-lightsource and the appropriate bands were cut using the x-tracta™ Gel Extractor tool (Promega). Gel purification was carried out using the Macherey-Nagel Nucleospin Gel and PCR Clean-up Kit.

Cloning

Restriction digest

For restriction of vector and insert DNA approximately 1000 ng purified DNA, 5 µL 10x buffer and 1 µL of each of the two restriction enzymes were mixed in a 1.5 mL reaction tube. The amount of DNA used depended on the size of the desired fragment. Water was added to a final volume of 50 µL. Restriction was carried out at 37 °C for 1 h. The reaction was stopped by inactivating the enzymes at 80 °C for 20 min.

• PstI/EcoRI HF: NE 2.1
• PstI/SpeI HF: NE 2.1
• PstI/XbaI: NE 3.1
• all other combinations: NEB CutSmart Buffer

Dephosphorylation

For the desphosphorylation of vector DNA 5.56 µL Antarctic Phos Reaction Buffer (NEB) and 0.5 µL Antarctic Phosphatase (NEB) were added to the digestet DNA and mixed. DNA was incubated at 37 °C for 60 min. After 30 min of incubation 0.5 µL Antarctic Phosphatase was added to the DNA and mixed.

DNA Purification

After desphosphorylation the vector DNA was purified using the Macherey-Nagel Nucleospin Gel and PCR Clean-up Kit.

Ligation

For ligation of the constructed plasmid 50 ng vector DNA, 3 times as much insert DNA, 2 µL T4 Ligase Buffer (NEB), 0.5 µL T4 Ligase (NEB) and water added to a final volume of 20 µL were mixed in 1.5 mL reaction tube. Ligation was carried out at 16 °C over night. The reaction was stopped by inactivating the enzymes at 65 °C for 20 min.

Transformation of chemocompetent cells via heatshock

One -80 °C glycerol stock of chemocompetend cells was thawed on ice. 10 µL of the ligated DNA was prepared in a 1.5 mL reaction tube. 50 µL of the cells were added and incubated for 20 min on ice. The mixture was then heat shocked at 42 °C for 1 min and cooled on ice for 2 min. 1 mL SOC-medium was added and the cells were incubated for 1 h at 37 °C while shaken at 600 rpm. 100 µL of the cell suspension were plated on a 2xYT agar plate with the according antibiotic (chloramphenicol or ampicillin) and incubated at 37 °C over night.

Electroporation

One -80 °C glycerol stock of chemo-competent cells was thawed on ice. 100 pg of purified DNA were prepared in a 1.5 mL reaction tube. 50 µL of the cells were added to the DNA and gently mixed. The cells were transferred into a precooled electroporation cuvette. Electroporation was carried out at 1.8 kV for 5.6 ms. The cells were resuspended in 1 mL SOC-medium, transferred into a 2 mL reaction tube. The cells were incubated at 37 °C and shaken at 600 rpm for 1 h. 100 µL of the cell suspension were plated on a 2xYT agar plate with the appropriate antibiotics and incubated at 37 °C overnight.

Expression of the His-constructs

50 ml 2xYT over night cultures of cells expressing HIS-tagged subunits of the MMO were inoculated from Glycerinstocks. After 24 h a main culture of 500 ml 2xYT-Ca-Glucose-Medium was inoculated to a starting OD₆₀₀ of 0,1 and cultivated at 37 °C and 250 rpm. Induction to a concentration of 100μM IPTG between OD 0,4-0,6. After induction the culutures were cultivated at 30 °C. 1mL of an arabinose solution (200 mg/mL) was added after 2 h and 4 h. After 6 h and 24 h after induction with IPTG 250 ml of the cells were harvested, centrifuged (13,000 g, 10 min) and the supernatant was discarded. The expression of the HIS-tagged subunits of the MMO was analyzed by using SDS-PAGE and Coomassie or Immunostaining respectively.

Expression of the mmo-construct

5 ml 2xYT over night cultures of cells expressing the MMO were inoculated from Glycerinstocks. After 24 h a main culture of 50 ml 2xYT-Ca-arabinose (4 mg/mL)-Medium was inoculated to a starting OD₆₀₀ of 0,1 and cultivated at 37 °C and 250 rpm. Induction to a concentration of 100μM IPTG between OD 0,4-0,6. After induction the culutures were cultivated at 30°C. 1mL of an arabinose solution (200 mg/mL) was added after 2 h and 4 h. The complete culture was harvested after 6 h.

Isolation of the soluble and inclusion fraction

The pellets harvested after 24 hours after induction with IPTG was resuspended in 5 ml PBS each. The cells were disrupted by sonification for 1 min and afterwards centrifuged for 15 min (800 g , 4 °C). The supernatant was transferred into a new 15 ml falcon and centrifuged for 30 min (6000 g, 4°C). The obtained supernatant contains MMO-subunits which are solubly produced. The pellet obtained after the first centrifugation step was resuspended in 10 ml PBS and again centrifuged for 15 min (6000 g, 4 °C). After the supernatant has been discarded this step has been repeated (resuspension in 10 ml PBS and centrifugation step). After the supernatant has been discarded the pellet was then resuspended in 2 ml PBS 8 M Urea added. The suspension was then transferred into a 2 ml Eppendorf tube and centrifuged for 20 min (11000 g, 4 °C). The obtained supernatant contains MMO-subunits which are produced in inclusion bodies.

The soluble fraction and inclusion body fraction was stored at -20 °C and later analysed using SDS-PAGE and Coomassie or Immunostaining respectively.

SDS-Page

The samples were mixed with 1x Laemmli Buffer with beta-Mercaptoethanol and heated for 10 min to 95 °C. The Gel was loaded with 10 μl of the prepared samples and run at 200 V for 50 min.

Coomassie Staining

After the SDS-PAGE has been executed the gel is put into a glass petridish filled with Coomassie Brilliant Blue coloring suspension and boiled up for a couple of second. The gel was incubated in the coloring suspension on a rocker for 10 min until the coloring was sufficient. Afterwards the gel was destained using destaining suspension and again boiled up for a couple of seconds and incubated on the rocker for an hour. The gel was stored in water until it was fully destained.

Western Blot

Blotting with Trans-blot SD semi dry transfer cell:
Order from top to bottom:

1. filter paper
2. gel
3. blot-membrane
4. filter paper

Activation of blot-membrane with MeOH (100 %). Filter paper are moistened with running buffer
Run of blot: 15V for 30 min

Immunostain

After having executed the Blot, the membrane was blocked with milk powder for 1 hour on a rocker. After 2 washing steps with PBS (0,1 %) (1x short, 1x for 5 min) the membrane was incubated for an hour in the first antibody solution on the rocker (Qiagen Anti Penta his (mouse) 1:2000). Again there was a washing step with PBS (1x short, 1x for 5 min). The membrane was then incubated for an hour in the second antibody solution (Dianova Goat anti Mouse IgG AP-conjugated 1:30.000, Predilution of antibody already 1:30) on the rocker. After the incubation the membrane was washed 3 times with PBST (2x short, 1x for 10 min) and twice washed with substrate buffer (1x short, 1 x for 5 min). The blot is then incubated in substrate buffer with NBT and BCIP (1: 100 in substrate buffer) until bands are visible (1-20 min). Afterwards the blot was washed twice with water (1x short, 1x for 5 min) and dried between two filterpapers.

sMMO activity assay

50 mL bacterial suspension was cultured as described in “Expression of the mmo-construct”. The culture was diluted to OD₆₀₀ 4 and centrifuged and washed twice with 0,9 % saline solution. The bacterial pellet was resuspended in 50 mL NMS-Medium. The methane measurement was performed in a modified anaerobic jar. Therefore the solution was placed into the anaerobic jar and was treated with methane and compressed air in order to obtain an atmosphere with 2 % methane. The methane concentration was detected with an “MQ-4 Semiconductor Sensor“ for 10.000 sec. A converted anaerobic jar in combination with a methane sensor was used for measurements. (https://static.igem.org/mediawiki/2014/0/09/TU-BS_sensor_sensor-in-pot.jpg) Figure 2: Methane sensor in the anaerobic jar For the measurements we used the sensor MQ-4 Semiconductor Sensor for Natural Gas by Henan Hanwei Electronics. Its function is based on SnO2 whose low conductivity rises with higher concentrations of combustible gases like methane or propane. This sensitivity for methane and its low costs made it perfect for our do it yourself methane measuring system. The data from the sensor were processed by a Sainsmart nano V3.0 device, a smaller variant of the Arduino board. https://static.igem.org/mediawiki/2014/5/59/TU-BS_sensor_methanesensor.jpg Figure 3: The methane sensor MQ-4 used in our experiment (left) https://static.igem.org/mediawiki/2014/e/e1/TU-BS_sensor_arduino.jpg Figure 4: The Sainsmart Arduino Nano v3 Board for processing the data coming from the MQ-4 sensor. To get the right methane concentration for each experiment we used flow controllers to set up the respective flow rates for methane, air and carbon dioxide which were then conducted through the anaerobic jar for 10 minutes until a homogenous methane atmosphere was formed. https://static.igem.org/mediawiki/2014/c/c6/TU-BS_sensor_gas-composition.jpg Figure 5: Tubing system and flow controllers to generate a desired methane atmosphere When the atmosphere needed was generated and all vents were closed the anaerobic jar was placed in an incubator set to correct temperature. For some experiments a magnetic stirrer was also placed in the incubator to keep the homogenity of the culture and the atmosphere in the jar. The sensor was connected to the Arduino board which was then plugged into the PC outside the incubator via USB cable. https://static.igem.org/mediawiki/2014/e/eb/TU-BS_sensor_incubator.jpg Figure 6: Complete setup of our methane measuring system We used a modified version of the Serial Client programmed by Michael Osthege from iGEM Aachen converting the processed signal of the Arduino board into a visual graph and a exportable table file. (https://2014.igem.org/Team:Aachen/Collaborations/Methane_Sensor). For the experiments, we filled the jar with a defined atmosphere of 2% methane in 98% air until the sensor displayed a stable methane concentration which was then set as 2%.The subsequently measured data were considered in relation to this value. Measurements were started after the sensor had adapted to humidity and temperature in the anaerobic jar. After the measurement the jar was filled with compressed air to define a zero-point.

Entrapment of E. coli

50 mL of an overnight culutre was pelletized. The pellet was washed twice in 0,9 % saline solution resuspended and diluted to OD₆₀₀ 4 in 10 mL 0,9 % saline solution. 1,5 g alginate was solved in 40 mL ddH₂O and was briefly boiled up. The alginate solution was mixed with all requiered antibiotics and arabinose (4 mg/mL). The whole solution was cooled and mixed with 10 mL bacterial 0,9 % saline solution. Beads were droped with a transfer pipette into a 0,05 M CaCl₂ solution. The CaCl₂ solution was decanted after 10 min.

Counting of cells with Thoma counting plate

Cell culture is placed on the plate and covered with a cover plate.
Sizes of the counting plate: depth: 0.02 mm area: 0.0025 mm². (Volume of a C-field: 0,0005 mm³ ).
The number of cells per mL was calculated appropriatly.

Cultivation on rumen fluid agar plates

We prepared agar plates with different concentrations of sterile filtrated rumen fluid, each with and without supplementation of glucose (100 mM). One half of each of these plates was inoculated with unsterile rumen fluid and the other one with E. coli. The plates were incubated over night at 37°C.