Physiology

Laboratory Notebook

15-08-11

First we examined the growth performance of #IGEM# (gene mdh in pSB1A30, T7 promoter!)

Prepare M9+Amp plates.

Plate #IGEM# & #6DA3# on M9+A plates.

Do LB overnight cultures of #IGEM# & #6DA3#.

Order 2 flowerplates with seal foil from Juelich.

15-08-12

Goal

make a nice growth curve
establish the procedure

Inoculation (make sure that cultures are comparable)

spin down 4 ml of overnight culture into one 2 ml tube
resuspend in 500 µl of sterile medium
measure the OD in a 1:10 dilution
add medium to adjust the OD to 5
in 50 ml falcons, prepare LB+A+MeOH+IPTG
- 1.5 mM IPTG (150 µl stock)
- 120 mM methanol (243 µl => 0,486 %vol; => 120 mM (like in Juelich-paper))
inoculate 50 ml medium with 0.5 ml of cells at OD=5

Cultivation

incubate the culture in shake flasks, V_R=500 ml, V_L=50 ml, T=37 °C, n=250 rpm

Sampling

in 1 h intervals, take 200 µl to measure in microplate reader measure OD in a MTP

Run 4 maincultures. 1 with LB+A+IPTG+MeOH #IGEM#; 1 with LB+A+IPTG+MeOH #6DA3#; 1 with LB+A+IPTG #IGEM#; 1 with LB+A+IPTG #6DA3#. (methanol in 2 shake flasks from the beginning)

Measure each hour in transparent microtiterplate in one row from left to right (8 measurements per microtiterplate A-H). Layout 3xIGEM normal, 3x 6DA3 normal, 3x IGEM MeOH, 3x 6DA3 MeOH in a row.

Measurement at 11:00 row A; at 12:00 row B; ... Do overnight cultures of #IGEM# & #6DA3# both in LB+A and M9+A (for biolector experiment).

15-08-13 biolector MDH function

Do biolector experiment with 3 different MeOH concentrations: 120 mM, 240 mM and 360 mM.

We prepare each well condition combination separately in 1 ml volume in a tube. Overnight cultures are centrifuged and diluted to OD 5. Used cultures are #IGEM# & #6DA3#.

LB blank: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp
M9 blank: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp
LB MeOH 120 mM blank: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 4.86 µl MeOH
M9 MeOH 120 mM blank: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 4.86 µl MeOH
LB MeOH 240 mM blank: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 9.72 µl MeOH
M9 MeOH 240 mM blank: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 9.72 µl MeOH
LB MeOH 360 mM blank: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 14.58 µl MeOH
M9 MeOH 360 mM blank: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 14.58 µl MeOH
IGEM LB ohne: 1 ml LB media, 3 µl IPTG, 1 µl Amp, 10 µl culture of OD5
IGEM M9 ohne: 1 ml M9 media, 3 µl IPTG, 1 µl Amp, 10 µl culture of OD5
IGEM LB MeOH 120 mM: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 4.86 µl MeOH, 10 µl culture of OD5
IGEM LB MeOH 240 mM: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 9.72 µl MeOH, 10 µl culture of OD5
IGEM LB MeOH 360 mM: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 14.58 µl MeOH, 10 µl culture of OD5
IGEM M9 MeOH 120 mM: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 4.86 µl MeOH, 10 µl culture of OD5
IGEM M9 MeOH 240 mM: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 9.72 µl MeOH, 10 µl culture of OD5
IGEM M9 MeOH 360 mM: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 14.58 µl MeOH, 10 µl culture of OD5
6DA3 LB ohne: 1 ml LB media, 3µl IPTG, 1µl Amp, 10 µl culture of OD5
6DA3 M9 ohne: 1 ml M9 media, 3µl IPTG, 1µl Amp, 10 µl culture of OD5
6DA3 LB MeOH 120 mM: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 4.86 µl MeOH, 10 µl culture of OD5
6DA3 LB MeOH 240 mM: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 9.72 µl MeOH, 10 µl culture of OD5
6DA3 LB MeOH 360 mM: 1 ml LB media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 14.58 µl MeOH, 10 µl culture of OD5
6DA3 M9 MeOH 120 mM: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 4.86 µl MeOH, 10 µl culture of OD5
6DA3 M9 MeOH 240 mM: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 9.72 µl MeOH, 10 µl culture of OD5
6DA3 M9 MeOH 360 mM: 1 ml M9 media, 3 µl IPTG (from 1000x stock), 1 µl Amp, 14.58 µl MeOH, 10 µl culture of OD5

Well volume: 150 µl (see plate-layout).

15-08-14

Take wellplate from biolector (BioVT) in the evening, seal it and freez it. no expected growth curve

The wellplate got discarded on the 22nd of August because we already got better samples for MeOH quantification via HPLC.

15-08-20

Growth of control and AP19.POLY on M9 with 1.25 M methanol (Shake flasks experiment)

Precultures (Overdays): In the morning, two 5 ml M9+K+40mM glucose cultures were inoculated with 100 µl LB overnight cultures of pSB1KRDP (#RP1O#) and AP19.POLY in pSB1KRDP (#AW9K#).

Carbon-limited M9 medium with methanol was prepared (8mM glucose, 1.25 M methanol).

In the evening, the cultures were centrifuged down and resuspended in 500 µl of fresh medium. OD was measured in 100x dilution and medium was added to adjust both to an OD of 3.75. Two shake flask cultures were prepared and inoculated with 1 ml to an initial OD of 0.15 (V_R=250 ml, V_L=25 ml, n=250 rpm, T=37 °C).

15-08-21

Growth of Control, Mdh expression plasmid and AP19.POLY on LB with 1.25 M methanol (Shake flasks experiment)

Used clones are control #RP1O#, "Mdh expression" #IGEM# & AP19.Poly #AW9K#. (#IGEM# has a T7 promoter and AmpR)

The overnight cultures were centrifuged down and resuspended in 500 µl of fresh medium. The OD was measured in a 100x dilution and medium was added to adjust to an OD of 3.75. Three shake flask cultures were prepared (Antibiotic and IPTG ones) and inoculated with 1 ml to an initial OD of 0.15 (VR=250 ml, VL=25 ml, n=250 rpm, T=37 °C).

next day: HPLC samples were taken to figure out the end concentration of MeOH in the media.
run SDS gel

Growth experiment in Biolector with different MeOH conc. in M9 C-lim media

Plate Layout 48 well flower plate

AP19.POLY (P)

MDH in pSB1A30 (M)

pSB1KRDP (C)

Blanc M9 medium (B)

Methanol Concentrations:

0 M (0 %)
0.75 M (3.04 %)
1.25 M (5.06 %)
1.6 M (6.48 %)

plate layout:

	1	2	3	4	5	6	7	8
A	C0	C0	C0	C0	C0.75	C0.75	C0.75	B0
B	C1.25	C1.25	C1.25	C1.25	C1.6	C1.6	C1.6	B0
C	M0	M0	M0	M0	M0.75	M0.75	M0.75	B0
D	M1.25	M1.25	M1.25	M1.25	M1.6	M1.6	M1.6	B0.75
E	P0	P0	P0	P0	P0.75	P0.75	P0.75	B1.25
F	P1.25	P1.25	P1.25	P1.25	P1.6	P1.6	P1.6	B1.6

centrifugation in 15 ml falcon and resuspension with 1mL M9 (0 % MeOH, 40 mM)
measure OD in 1:100 dilution and adjust to OD x
from this dilution, do another 1:100 dilution in Eppis
inoculate falcons with different MeOH concentrations with x µl
750 µl in each well
shaking at 900 rpm

15-08-22

The Biolector experiment was stopped after 22 h and all wells were prepared for HPLC analytics (Sample Nr. D8,E8,F8 & F5, F6, F7 & B5, B6, B7 & D5, D6, D7 for triplicates are already in HPLC; rest is freezed).

3 samples of the LB media with MeOH shake flask experiment were also analysed by HPLC.
Some wells are also prepared for SDS-PAGE for the proof of protein expression.

15-08-23

Evening: Do overnight cultures of Mdh expression clones from masterplate.

Shake flask experiment (1. of the day)

run shake flask experiment of #RP1O# with three different MeOH concentrations

experiment started at 10:00

inoculated to OD 0.2

flask 1: #RP1O#, 25 ml LB, 25 µl K, 0 M

flask 2: #RP1O#, 25 ml LB, 25 µl K, 0.75 M

flask 3: #RP1O#, 25 ml LB, 25 µl K, 1.25 M

MeOH added after 2 h

HPLC samples:

t [h] after MeOH induction	HPLC sample?
0	yes
2	yes
4	yes
6	?
8	?
10	?

Shake flask experiment (2. of the day + night)

Grow polycistronic clone #AW9K# in LB media with different MeOH concentrations (0 M; 1,25 M; 1,6 M).

TO DO

repeat SDS gel of shake flask experiment samples (15-08-22)
SDS gel of Biolector experiment samples (15-08-22)
make plates and overday cultures of #IGEM#, #RP1O# and #AW9K# for further experiments
run shake flask experiment of #RP1O# with three different MeOH concentrations

15-08-24

continue shake flash experiment 2 from last night

three cultures are grown until stationary phase. We used 20 ml of these cultures to restart the cultivation in new flasks. We added methanol and water to an end concentration of 1,25 M MeOH.

Expected results: If the culture in flask 1 and 2 start to grow again it is proofed that E.coli uses Methanol as a carbon source.

Layout:

conti poly experiment in shakeflask

made by iTS; start 15:15.

24 ml LB+K added to old shake flask No.3 from last night experiment (which was 1.25 M MeOH before) #AW9K# is the cultivated clone. 2 ml MeOH are added (8.3 vol %; 2.0571 M MeOH).

start OD is 0.382 (at 15:15)

10:50 => Triplett OD 0.59; 0.54; 0.60.

17:45 => Triplett OD 0.77; 0.73; 0.66 & not diluted 0.515.

15-08-25

Biolector experiment

For each strain (AP19 Poly in pSB1KRDP from #AW9K# and pSB1KRDP control from #RP1O#), one 10 ml flask preculture was inoculated to OD 0.2 at 8:50 with 0.5 ml of overnight. The flower plate was inoculated to an OD of 0.3 with 20 µl of cells.

Plate layout:

	1	2	3	4	5	6	7	8
A	C0	C0	C0	C0	P0	P0	P0	P0
B	C0.75	C0.75	C0.75	C0.75	P0.75	P0.75	P0.75	P0.75
C	C1.25	C1.25	C1.25	C1.25	P1.25	P1.25	P1.25	P1.25
D	C1.6	C1.6	C1.6	C1.6	P1.6	P1.6	P1.6	P1.6
E	C2.0	C2.0	C2.0	C2.0	P2.0	P2.0	P2.0	P2.0
F	C2.5	C2.5	C2.5	C2.5	P2.5	P2.5	P2.5	P2.5

(C=control strain with pSB1KRDP, P=AP19.poly in pSB1KRDP)

Shake flask experiment (AP19 Poly with three different MeOH concentrations)

flask 1: AP19 Poly, LB+K+ 0.75 M MeOH

flask 2: AP19 Poly, LB+K+ 1.25 M MeOH

flask 3: AP19 Poly, LB+K+ 1.6 M MeOH

80 ml preculture LB+K
- inoculated to OD 0.1 at 9:23

OD values:

time	runtime	OD
9:23	0	0,11
10:23	1	0.20
11:23	2	0.39

Experiment in three 250 ml shake flasks started at 11:25
Methanol was added at 13:15 (runtime: 1.8 h). OD: 0.951(1) 0.969(2) 0.947(3)

START AGAIN

this experiment was started because of non sufficient growth curves again in the evening
MeOH induction at 1:25
- OD: 0.75 M= 1.402; 1.25 M= 1.392; 1.6 M= 1.399
- a.u.: 0.75 M= 121.416; 1.25 M= 118.142; 1.6 M= 113.791
experiment stopped at 11:00
- OD: 0.75 M= 6.62; 1.23 M= 4.98; 1.6 M= 3.12
SDS Page and HPLC samples were taken

15-08-26

At 0:15 in the morning, we inoculated M9+K+40 mM glucose precultures (V_R=100 ml, V_L=10 ml) with different volumes (10, 30, 80, 160 µl) of previously grown LB+K preculture of AP19.Poly in pSB1KRDP.

At ca. 13:00 three shake flasks were inoculated with 2 ml of preculture with an OD of 2.25.

Methanol (0, 0.75 and 1.25 M) was added to the flasks and the OD was determined to be 1.51, 1.6 and 1.66 respectively. (HPLC and SDS samples were taken too.)

15-08-28

At 23:00 we inoculated M9+K+40mM glucose precultures (V_R=100 ml, V_L=10 ml) with different volumes (20, 40, 80, 160 µl) of previously grown LB+K preculture of AP19.Poly in pSB1KRDP.

15-08-29

The main culture flasks were put into the shaker for pre-warming and blank measurement.

At 10:15 the highest preculture-OD was 0.94 (Note: Next time inoculate 50, 100, 160 and 320 µl)

At 11:50 the highest preculture-OD was 1.56

Inoculated at 12:50 with 2 ml of preculture with an OD of 2.29

15-08-30

Endpoint OD of shakeflask experiments from yesterday:

MeOH conc	OD 1	OD 2	OD 3	OD
0M	4.66	4.45	3.65	4.25 ± 0.44
0.75M	4.48	4.42	4.23	4.37 ± 0.11
1.25M	4.46	3.95	3.94	4.12 ± 0.24

15-09-01 Mdh Assay (in vitro)

The Mdh Characterization started now in parallel.

15-09-04

Compairability test

with #IGEM# in LB+K
OD end values vs arbitrary units:
- flask 1: 8.3 | 8.8 | 8.3 (8.47 +- 0.24)
- flask 2: 7.7 | 7.8 | 7.7 (7.73 +- 0.05)
- flask 3: 7.9 | 7.5 | 7.6 (7.67 +- 0.17)

AP19 Poly shake flask in LB

#AW9K# precultures in LB
- inoculated with 1 ml to OD 0.3
- MeOH inoculation (check annotation)
  - flask 1: 0.25 M MeOH
  - flask 2: 0.5 M MeOH
  - flask 3: 0.75 M MeOH

end OD triplicate values
- flask 1: 6.0 | 6.3 | 6.5
- flask 2: 5.9 | 6.3 | 6.2
- flask 3: 4.8 | 5.1 | 5.4

HPLC and SDS samples in -20 °C freezer

15-09-05

shake flask experiment(36 flasks)

Started at 14:16.