Labeling Experiment

Laboratory Notebook

General Design

We got 5 g ¹³C with full deuterium labeled methanol and 1 g normal ¹³C labeled methanol for our experiment (Product Numbers: 293865 Aldrich Methanol-13C,d4 99 atom % ¹³C, 99.5 atom % D and 277177 Aldrich Methanol-¹³C, 99 atom % ¹³C ).

We used reactor 5,6,7 & 8 for our ¹³C labeling experiment. We adjust the pH to 7. The oxygen sensor was adjust to 30 % solved oxygen.

Reactor 5: (#AW9K#) gets 200 ml M9 media with 0.7 M methanol therefore we inject 2.73 ml full labeled methanol and 3.34 ml non labeled methanol => the labeled methanol relatives are 45.3 %.

Reactor 6) (#AW9K#) gets 200 ml M9 media with 0.3 M methanol therefore we inject 2.51 ml full labeled methanol.

Reactor 7) (#IGEM#) gets 200 ml M9 media with 0.15 M methanol therefore we inject 1.23 ml ¹³C labeled methanol.

Reactor 8) (#IGEM#) gets 200 ml M9 media with 0.15 M methanol therefore we inject 1.22 ml normal non-labeled methanol.

reactor	strain	media	methanol conc.	MeOH volume
5	#AW9K#	M9 40 mM Glucose, Kanamycin	0.75 M	2.73 ml of ¹³C-MeOH with four deuterium atoms; 3.34 ml of normal MeOH
6	#AW9K#	M9 40 mM Glucose, Kanamycin	0.31 M	2.51 ml of ¹³C-MeOH with four deuterium atoms
7	#IGEM#	M9 40 mM Glucose, Ampicillin	0.15 M	1.23 ml of ¹³C-MeOH
8	#IGEM#	M9 40 mM Glucose, Ampicillin	0.15 M	1.22 ml of normal MeOH

The samples for analysis of cytosolic metabolites were taken after one doubling time.

The analysis will be done by mass spectrometry of cytosolic metabolites: fructose-bis-phosphate, glucose-6-phosphate and ribose-5-phosphate, DHAP, Xylulose-5-phosphate, Fructose-6-phosphate, E4P, 2Phosphoglycerat/3Phosphoglycerate, S7P, AMP & ADP.

Procedure

Reactor 7 & 8 got 200 µl IPTG at an OD of about 0.8 to induce the #IGEM# clones to express the methanoldehydrogenase.

At an OD of about 1.5 each reactor gets the planned volume of the related kind of methanol injected.

Reactor 7 is connected to an offgas-analyses device to detect ¹³C carbondioxid which may be produced via the detoxification system of E.coli because the strain #IGEM# in reactor 7 expresses the methanoldehydrogenase which form formaldehyde out of methanol.

A reactor is sampled three times at an OD of 3. Additionally the biovolume of each sample is measured with a coulter counter. These samples got directly freezed by adding 10 ml sample into just melting 60% methanol.

These cooled samples got centrifuged (-20 °C) (the supernatant was discarded) and the pellet was resuspendet via vortexing in 1:1 relationship methanol and chloroform according to the total biovolume.

The resuspendet pellets were put into an overhead-shaker in the -20 °C room for at least two hours to extract intracellular comonents.

The solution get centrifuged for 10 min and the supernatant get transfered into a syringe with filter (0.2 µm) to filter it sterile into a 1.5 ml tube.

The samples will be analysed by mass spectrometry (MS).

Expected MS-Results

3 different sugars are analysed: Fructose-bis-Phosphate; Glucose-6-Phosphate and Ribose-5-Phosphat.

Mass shift when ¹³C,d4 is assimilated (methanol is m+5, formaldehyde is m+3):
- all following sugars should have a shift of m+3 in the first cicle of the functional pathway.

Mass shift when ¹³C is assimilated (methanol is m+1, formaldehyde is m+1):
- all following sugars should have a shift of m+1 in the first cicle of the functional pathway.

A mass shift of up to m+6 is in the range of measured shifts.

Mass Shift explanation
This visualization of reactions with mass shifted molecules explains exemplary how we gain cytosolic metabolites with mass shifts starting from ¹³C methanol with four deuterium atoms.

Wetlab Notebook

15-09-06 Sunday

Preparation of M9 Media aliquote components.

15-09-07 Monday

Prepare M9 media for the whole experiment (4x 500 ml M9 40 mM Glucose)

Inocculate precultures from LB-agarplates into 10 ml M9 in 100 ml shake flasks. Start 13:20.

Preparation of 20x 60 % methanol stocks (30 ml in Falkons) and freeze them at -80 °C for quenching of samples (we should need 9 stocks).

15-09-08 Tuesday

Related to our MDH-Characterization experiments we decided to use BL21 DE3 as expression strain to do the labeling experiment.

Do in 50 ml shake flask cultures in M9. Therefore we devided the 10ml overnight cultures into two equal oculated shake flasks with 50ml M9 (40 mM Gluc). Incubation started at 13:00

Do two more M9 cultures of each clone to get clones in the exponential phase next day. Therefore we oculated 0.5 ml of #IGEM# flask II into a new one and 0.1 ml into an other one. From #AW9K# we oculated 1 ml and 0.5 ml from shake flask II into new media.

=> So we got four precultures for our following bioreactor experiments. The incubation of this new additional cultures started at 15:00.

Prepare the OD measurements for the next day.

We build up and autoclaved all four reactors (Reactor number 5,6,7 & 8) and filled them up with 200 ml M9 media with antibiotics.

The oxygen calibration was done over night to start in the next morning. The temperature of the reactors was set to 37 °C.

15-09-09 Wednesday

We choosed shake flask IV of the #AW9K# clone with an OD of 5.90 (measured in triplicate) to inocculate reactor 5 and 6 to a start OD of 0.3. Therefore we made a pellet out of 10.169 ml of the culture, washed it with 0,9%NaCl solution and pellet it again and resuspendet it in 2ml fresh M9 media. The inoculation was done with these 2ml cellsuspension.

We chosed shake flask II from clone #IGEM# with an OD of 5.43 (measured in triplicate) to inocculate reactor 7 and 8 to a start OD of 0.3. Therefore we made a pellet out of 11.050 ml of the culture, washed it with 0,9 % NaCl solution and pellet it again and resuspendet it in 2 ml fresh M9 media. The inoculation was done with these 2 ml cellsuspension.

All reactors are inoculated and started at 9:20.

realtime	experiment time	OD reactor 5 (AW9K)	OD reactor 6 (AW9K)	OD reactor 7 (IGEM)	OD reactor 8 (IGEM)	comment
10:20	1:00	0.346	0.333	0.371	0.371
11:20	2:00	0.281	0.270	0.282	0.288
12:30	3:10	0.298	0.292	0.319	0.328
13:20	4:00	0.350	0.350	0.428	0.415
14:05	4:45	0.493	0.508	0.683	0.645
14:25	5:05	0.427	0.442	0.641	0.598
14:50	5:30	0.55	0.60	0.91	0.77	measuring diluted 1:10 starting from now
14:59	5:39	-	-	+200 µl IPTG	+200 µl IPTG	IPTH 1000x stock
15:55	6:35	0.75	0.81	1.32	1.14
16:08	6:44	-	-	MeOH induction	-	Reactor 7 got ¹³C MeOH to reach 0.15 M
16:15	6:50	0.79	0.92	1.47	1.26
16:23	6:58	-	-	-	MeOH induction	Reactor 8 got MeOH to reach 0.15 M
17:15	7:50	1.08	1.21	1.96	1.64
17:26	8:01	-	MeOH induction	-	-	Reactor 6 got Deut.¹³C MeOH to reach 0.3 M
17:29	8:04	MeOH induction	-	-	-	Reactor 5 got Deut.¹³C MeOH and MeOH to reach 0.75 M
18:20	8:55	1.30	1.72	2.82	2.0
19:10	9:45	-	-	3.84 => sampling	-	3x10 ml sample for quenching were taken from reactor 7. Biovolume measured with coulter counter (dil. 1:2000) (of reactor 7).
20:30	11:05	2.3	3.6 => sampling	--	3.24	3x10 ml sample for quenching were taken from reactor 6. Biovolume measured with coulter counter (dil. 1:2000) (of reactor 6).
21:00	11:35	sampling	--	--	--	3x10 ml sample for quenching were taken from reactor 5. Biovolume measured with coulter counter (dil. 1:2000) (of reactor 5).

The arithmetic middel biovolume of the sample of reactor 5 is 1.418938766 mul/n sampled vol

The arithmetic middel biovolume of the sample of reactor 6 is 2.58966315 mul/n sampled vol

The arithmetic middel biovolume of the sample of reactor 7 is 2.714383377 mul/n sampled vol

(These values will be important the next day when MeOH/TE buffer and chloroform is added to the sample to extract the intracellular metabolits.)

Each 10 ml sample for metabolic analysis was taken in triplicates. Each was taken into 30ml just melted (ca. -50 °C) 60 % methanol solution to freeze the samples directly to have no metabolic activity any more (quenching).
The quenched samples get centrifuged for 10 min at -20 °C. The supernatant was discarded and the pellet is used the next day.

15-09-10 Thursday

We took 10 ml samples for Nash Assays of each reactor at 9:00 for further analyses. We centrifuged them and freezed the cell pellets and the media supernatant (see MDH Characterization).

We deinstall all reactors with all related components.

The freezed (-80 °C) labeling samples were used for extraction of intracellular comonents. Therefore the whole procedure was done at at least -20 °C in each preparation step. We added MeOH/TE Buffer depending on the biovolume to the falcons in a -30 °C methanolbath. The same amount of Chloroform was added too. The pellets got solved via vortexing several times in intervalls to keep the temperatute low.

The resuspendet pellets were put inro an overhead-shaker in the -20 °C room for at least two hours (start 10:59).

The solution get centrifuged for 10min and the supernatant get transfered into a syringe with filter (0.2 µm) to filter it sterile into a 1.5 ml tube.

Til here the work was done by members of our team (Jonas R. & Tobias S.) with instructions. All following metabolit data is generated by scientists from Forschungszentrum Juelich (Jannick Kappelmann). The interpretation of data was a collaboration with Jannick Kappelmann.

MS-Data of analytic standards

These are the average compositions of the analysed carbonhydrates. They serve as contole for natral compositions.

Fructose-biphosphate

Sample Name	Standard	FBP m.0.0	FBP m.1.0	FBP m.2.0	FBP m.3.0
150911_ZSW-markiert_100915_iGEM-s24	¹²C analytes	0.90267	0.0753519	0.0200073	0.00197117

Glucose-6-phosphate

Sample Name	Standard	G6P m.0.0	G6P m.1.0	G6P m.2.0	G6P m.3.0	G6P m.4.0
150911_ZSW-markiert_100915_iGEM-s24	¹²C analytes	0.917202	0.068935	0.0124054	0.000869739	0.000588146

Ribose-5-phosphate

Sample Name	Standard	R5P m.0.0	R5P m.1.0	R5P m.2.0
150911_ZSW-markiert_100915_iGEM-s24	¹²C analytes	0.925831	0.0638775	0.0102916

Plot of natural FBP, G6P & R5P as control
This figute shows the natural mass shift composition of the measured metabolites.

MS-Data - Reactor 5 (0,75 M with 45,3 % ¹³C,d4)

Fructose-bis-Phosphate values: