Characterization

Laboratory Notebook

15-08-13

electroporation of glgC (#VMN3#) in cured BL21 ΔglgX and cured BL21 ΔglgP
make two 250 ml main cultures (M9 medium) of both wild type BL21 and cured BL21 ΔglgP at 3:15 pm
- cultures grow for 20 h

15-08-14

centrifuge cultures after 20 h (11:15 am) and freeze pellet
master plate of BL21 ΔglgX with glgC (BL21 ΔglgP with glgC did not grow)
repeat electroporation of glgC in pSB1A30 into BL21 ΔP (#RX39# as control)
heat shock of glgC in pSB1A30 into BL21

15-08-15

master plate of glgC in BL21 ΔP and glgC in BL21
overnights of glgC in BL21, glgC in BL21 ΔglgP and Bl21 ΔglgX

15-08-16

cryo cultures
Plasmid Preparation and test digest of glgC plasmid

type	cryo	plasmid for test digest	tube no in digest	chosen for characterization
BL21 WT glgC in A30 #1	#KW1B#	#ZDHM#	8
BL21 WT glgC in A30 #2	#YHRZ#	#H4M4#	9
BL21 WT glgC in A30 #3	#XAMZ#	#DTRB#	6
BL21 ΔglgX glgC in A30 #1	#1PP9#	#XHZC#	5	yes
BL21 ΔglgX glgC in A30 #2	#NN4O#	#YSHR#	3
BL21 ΔglgX glgC in A30 #3	#NDAB#	#CPPQ#	1
BL21 ΔglgP glgC in A30 #1	#ERWT#	#FAEW#	2	yes
BL21 ΔglgP glgC in A30 #2	#QXQR#	#Y4LX#	4
BL21 ΔglgP glgC in A30 #3	#QHCH#	#TTT3#	7

15-08-17

test glgC in BL21 ΔglgX, glgC in BL21 ΔglgP, and BL21 wild type
- started 5 ml LB- precultures at 14:00
- started 10 ml M9 precultures at 18:30

first OD measurement of M9 preculture at 19:00

	1	2	3
BL21 wild type	0.018	0.03	0.03
BL21 ΔX + glgC	0.02	0.017	0.02
BL21 ΔP + glgC	0.007	0.012	0.013

15-08-18

ΔglgX + glgC and ΔglgP + glgC growth experiment
- M9 cryo cultures #QRYS# and #TY1V#
- inoculated main cultures (50 ml) to OD 0.2 at 8:30 am
- for every sample: OD, pellet with 10⁹ cells and supernatant for HPLC

ΔglgX + glgC and ΔglgP + glgC growth experiment
Taking into account different OD starting points, it was observed that ΔglgP + glgC grew to an OD of 5, equally as the wild type. ΔglgX + glgC grew faster but only to an OD of 3.8.

15-08-19

test for rapid determination of glucose and nitrogen
- glucose was not depleted in stationary phase (approximately 35-55 mmol)
- nitrogen was not depleted in the stationary phase
- new M9 medium will contain less nitrogen
growth curve shows that BL21 ΔglgX has a shorter lag phase than the control and dies earlier
taking into account the different starting OD, ΔglgP and the wild type grow equally

made 5 ml LB cultures of Bl21 ΔglgX, BL21 ΔglgP and BL21 WT at 1 pm
inocultated an 10 ml M9 preculture at 6 pm

15-08-20

growth experiment with BL21 ΔglgX, BL21 ΔglgP and BL21 WT
- inoculated with OD 0.5
- took OD samples, samples for glycogen quantification and HPLC
- cultures stopped growing at OD 1
  - test showed that nitrogen was depleted after 9 hours and growth curve indicates that it had already been depleted earlier
  - for future experiment, the amount of nitrogen in M9 will be raised

15-08-21

made 5 ml LB cultures 11:30 am
inoculated 10 ml M9 precultures at 6 pm
M9 medium now contains 6.6 mM nitrogen ans 40 mM glucose

15-08-22

inoculated 25 ml main culture at 9:30 am (wild type, ΔglgX + glgC, ΔglgP + glgC)
- measured OD with Aquila Biolabs "Cell Growth Quantifier" (CGQ)

ΔglgX + glgC and ΔglgP + glgC growth experiment online
This growth curve resulted from an online OD measurement with Aquila Biolabs "Cell Growth Quantifier"(CGQ). We observed that ΔglgX and ΔglgP showed the same growth behavior as in the previous experiment where the OD was measured manually. ΔglgX has a shorter lag phase but dies earlier at a lower OD than the wild type and ΔglgP.

purified glycogen samples from this experiment: #Z19V# and #BV1E#

15-08-23

used protocol for glycogen extraction from the glycogen kit to analyze cultures wild type and ΔglgX + glgC in pSB1A30
let samples dry over night
5 ml LB overnight cultures of BL21 ΔX, BL21 ΔP and BL21 WT (6 pm)

15-08-24

remaining ethanol from glycogen purification is evaporated by vacuum centrifugation
1 ml of LB overnight transferred in 5 ml of new LB
25 ml M9 main cultures inoculated at 4:15 pm
measure OD with Aquila Biolabs online measurement tool
duplicates inoculated, samples taken every 2 hours for HPLC

15-08-25

standard curve from 15-08-20 resulted in formula y/8168,2=x

Y=the detected emission and X=the amount of glycogen in µg

	amount of glycogen (dilution 1:25)	aount of glycogen (dilution 1:50)
BL21 wild type	0.564 µg	0.472 µg
BL21 ΔX + glgC	0.463 µg	0.260 µg

5ml LB cultures for growth test: BL21 WT, BL21 + GlgC, Bl21 delta X +GlgC
extracted glycogen usinf the Glycogen Kit from BL21, BL21 ΔglgX and BL21 ΔglgP (from 15-08-20)
- purification failed due to human error

15-08-26

growth test of BL21 Gold wild type, BL21 Gold ΔglgX + glgC and BL21 Gold + glgC
- inoculate main cultures at 11.45 with OD 0.17 (25 ml of M9 medium 6.6 mM nitrogen)
- induced after 4 h 32 min (appr. OD 2)
- measured OD with Aquila Biolabs online measurement tool
quantification of extracted glycogen BL21 WT, BL21 ΔglgX and BL21 ΔglgP
glycogen samples are: #9VFE# (WT), #ZVZ3# (X+C), #D9OT# (WT+C)

15-08-27

5ml LB cultures for growth test : BL21 WT, BL21 ΔglgP, Bl21 ΔglgX

15-08-28

BL21 WT, BL21 ΔglgP, Bl21 ΔglgX growth experiment
- 25 ml M9 main cultures inoculated to OD 0.2
- measured OD with Aquila Biolabs online measurement tool

ΔglgX and ΔglgP growth experiment online
Testing the growth of the single knockouts only showed that ΔglgX and ΔglgP had an identical growth behavior compared to the combination of each knockout with glgC. ΔglgP and the wild type grow very similarly while ΔglgX dies earlier. Aquila Biolabs CGQ was used for online measurement

iodine staining of cell pellets from first growth experiment (15-08-18; CDW approximately 0.4 mg)
- we used modiefied version of Lugol's iodine (5 mM I2 and 5 mM KI in water)
- ΔglgP + glgC is stained more than the WT

acid hydrolysis of WT and ΔglgX + glgC samples from 15-08-22
made M9 overnight cultures of BL21 Gold to test the pre-extraction with glycogen kit

15-08-29

glycogen extraction of BL21 WT, BL21 ΔglgX, BL21 ΔglgP
- used 21 ml of (adjusted to an OD of ~2.4)
- iMH and iMB exceeded the protocol and centrifuged one more time, resuspended the pellet in water, centrifuged again and used the supernatant
- the cell pellet was also stored
glyocogen extraction of two BL21 WT overnights to test the purification protocol
- used 4 ml of OD 1.83
for acid hydrolysis: evaporation of HCl under the hood at 85 °C
5 ml LB precultures of BL21 WT, BL21 ΔglgX + glgC and BL21 ΔglgP + glgC
- these cultures are inculated again to purify glycogen with samples that are adjusted to the same OD
- samples will be purified, crude glycogen dissolved in water and centrifuged
- supernatant will be used for acid hydrolysis
- glucose then quantified with glycogen assay kit and HPLC

15-08-30

evaporation of ethanol and water in vacuum centrifuge for both WT samples
for samples of iMB/ iMH, water samples were adjusted to pH 3
- then ethanol was added to the sample
- the pellet with cell material were dissolved in water and adjusted to pH 3
- ethanol was also added to these samples

glycogen samples of acid hydrolysis (Bl21 WT, BL21 ΔglgX + glgC from 15-08-22) were quantified with the glycogen kit
- attention: samples were not adjusted to the same OD
- glucose rapid determination showed that there is no glucose in the BL21 ΔglgX + glgC sample!!!

inoculated 25 ml M9 main culture at 2:40 pm

time	OD WT	OD ΔglgX + glgC	OD ΔglgP + glgC
0'10"	0.127	0.186	0.147
2'00"	0.592	0.663	0.528
4'10"	1.35	1.36	1.23
4'55" (induction)	2.31	1.96	1.74
18'00"	5.77	5.99	6.74

sample	volume (µL) WT	volume (µL) ΔglgX + glgC	volume (µL) ΔglgP + glgC
0	1000	683	864
1	215	192	241
2	94	93	103
3	55	65	73
end	22	21	19

plated #CL6W#, #WHPX# and #ZEYD# on LB-
made 10 ml M9 overnight of BL21 wild type

15-08-31

precultures of #CL6W#, #WHPX# and #ZEYD# (from LB- plate), plate #BXR1# (ΔglgP is in Bio6) on LB-
do iodine staining with samples from yesterday (from different growth phases)
- result: most glycogen is accumulated in the stationary phase

iodine staining with overday cultures (GlgA, GlgB, GlgC)
- adjust all to the same OD and resuspend in 50 µL of iodine solution
- result: all three samples (glgA, glgB and glgC) were darker than the wildtype, glgC was the darkest

with M9 culture of wild type:
- centrifuge the whole culture down in a 15 ml falcon
- resuspend in 5 ml NaCl (0.9 %) solution
- do a dilution series
- Centrifuge down
- stain with idodine solution
- pipette in well plate and measure absorbance

with overday cultures of WT, glgA, glgB, glgC:
- measure OD
- lowest OD: 1.8
- use 4 ml of this culture and calculate how much of other cultures do you need for equal cell number
- centrifuge cultures for 2 minutes
- discard supernatant and resuspend pellet in 300 µl iodine solution

plate BL21 Gold (DE3) WT, ΔglgX and ΔglgP on M9 and on LB- plates
to repeat absorbance measuremen: new 10 mL M9 overnights of Bl21 Gold (DE3) WT and ΔglgX (6.6 mM N)
- compare wild type to ΔglgX, because ΔglgX strain is expected to have no glycogen-> detect absorbance of iodine stained glycogen

5 mL LB overnights of NEB10β WT, ΔglgX, ΔglgP and ΔglgXP, on both normal LB and LB+40mM Glucose
new overnight of glgC in pSB1A30 to make a new cryo culture, because the old one did not grow so well

15-09-01

new cryo of glgC in pSB1A30 (#3OQZ#)
do overnights of GlgA, GlgB, WT Bl21, (in BL21: #FVNV# in H;B3) in LB with 20mM glucose

15-09-02

iodine staining (4 ml of OD 1.55, used 200 µl iodine solution)

Iodine staining glgA vs. wild type
Cultivated in LB + 20mM glucose, BL21 Gold + glgA stained distinctly darker than the wild type.

Dinitrosalicylic acid staining ( OD of WT was adjusted to the OD of the ΔglgX)
- 1 g of Dinitrosalicylic acid was dissolved in 20 mL 2 M NaOH, 30 g of Potassiumsodiumtartrate was added and the volume was adjusted to 100 mL with aqua bidest.
- 1:50, 1:25 and 1:20 dilutions of WT and ΔglgX were prepared; 50 µL sample volume. The same volume of Dinotroslicylic acid was added; total volume was 100 µL.
- the stained samples were heated for 10 minutes at 90 °C.
- OD 450--700 nm was measured immediatly after heating.

15-09-04

iodine staining of BL21 WT, BL21 glgA, BL21 glgB and BL21 ΔglgP in M9
- wildtype and ΔglgP are the darkest, GlgA is slightly lighter

15-09-05

HPLC

wash 10mL-overnights with NaCl, adjust to same OD
hydrolize cell pellet in 500ul HCl
prepare Glucose standard and samples for HPLC (sunday)

Iodine staining

prepare glucose standard (0.05 µg - 2 µg) no staining: the "glycogen standard" is most likely only glucose so it cannt be stained with iodine
adjust samples to same OD and do staining
- M9: WT, ΔglgP, glgB, glgA
- LB: WT, glgA, ΔglgP

Iodine staining ΔglgP vs. wild type
Cultivated in M9, BL21 Gold ΔglgP stained distinctly darker than the wild type.

15-09-06

Dinitrosalicylic acid staining (OD of WT, and ΔglgP was adjusted to the OD of the ΔglgX = 1,55)
1:100, 1:50, 1:25, 1:12.5, 1:6.25, 1:3.125, and 1:1.5625 dilutions of WT, ΔglgP, and ΔglgX were prepared; 50 µL sample volume. The same volume of Dinitrosalicylic acid was added; total volume was 100 µL. (prepare more than 100 µL per well per sample, since the solutions get evaporated in the 90 °C heater!)
- blank: 1:1 dilution of dinitrosalicylic acid in water.
- the stained samples were heated for 10 minutes at 90 °C
- the heated samples were cooled down in a 25 °C heater
- analysed in plate reader at 540 nm

15-09-07

iodine staining of BL21 WT, BL21 glgCAB #1 and #5
- adjusted to 4 ml at OD 1.64
- both glgCAB clones were darker than the wild type

File:Aachen glgCAB , WT.png

Iodine staining glgCAB vs. wild type
Cultivated in LB+20mM glucose, BL21 Gold + glgCAB stained distinctly darker than the wild type.

LB overnight cultures of BL21 glgCAB' in pSB1A30 (#1 and #5), BL21 glgC in pSB1A30 and BL21 wild type for iodine staining
make master plates and overnight cultures of #XULU# in BL21 Δ P

glycogen quantification of BL21 glgA, glgB, ΔglgP, ΔglgX and WT
- use 8 ml of each culture adjusted to OD 5.2
- centrifuge
- wash with NaCl two times
- resuspend pellet in 500-1000 µl 5 M HCl for acid hydrolysis over night (in glass vials, 105°C)

Dinitrosalicylic acid staining (OD of ΔglgX was adjusted to the OD of the WT = 2.8)
- blank: 1:1 dilution of dinitrosalicylic acid in water

15-09-08

pellets of WT Bl21, GlgCAB in C30 #1, #5 are adjusted to same OD (2.04) and were frozen to do iodine staining tomorrow (since GlgC did not grow) tomorrow: iodine staining (in dilutions for better colour comparison)
do masterplates of BL21 transformation of glgAB in pSB1C30
when do we get the HPLC?
make a calibration series of starch solutions and scan triplicates in a well plate
- series looks good, will be done with glycogen solutions tommorow
cryo cultures of #XULU# in BL21 Gold ΔglgP: #NKKA# #E4QF# #YNAZ# #R8LW# #B8FD#

15-09-09

sequencing did not work
do iodine staining with frozen pellets and GlgC
- the pellets were first solved in 50 ul water

Iodine staining glgCAB vs. wild type vs. glgC
Cultivated in LB+20mM glucose, BL21 Gold + glgCAB and BL21 + glgC stained distinctly darker than the wild type. It can be observed that GlgC is more blue which indicates more linear glycogen. GlgCAB #1 is very dark, brownish which suggests a higher frequency of branches. This could result from the influence of the branching enzyme.

iodine staining calibration
- prepare 50 µl glycogen solutions with 8, 4, 2, 1, 0.5, 0.25 and 0.125 g/l concentrations
- add 300 µl iodine solution to each solution
- scan triplets in well-plates

growth experiment of BL21 glgCAB #1 (#WCQY#), BL21 WT (#34CT#) and BL21 glgC (#OHES#)
- make 5 ml LB precultures

glycogen purification
- do precultures of BL21 glgCAB (#WCQY#), WT (#34CT#), ΔglgP (#FVNV#), ΔglgX (#C81O#) glgA (#K8DM#) and glgC (#VMN3#)

15-09-10

inoculate 25 ml LB + 20 mM glucose main culture for growth experiment of BL21 glgCAB #1, BL21 WT and BL21 glgC at OD 0.2
- start OD: C 0,236 CAB 0,19 WT 0,224
- induction: GlgC at OD 1,44, GlgCAB at 1,03
- use these cultures for SDS samples and iodine staining (1mL, OD=1,resuspend in 50 µl water first) in well plate

inoculate 25 ml LB + 20 mM glucose cultures with BL21 glgCAB, WT, ΔglgP, ΔglgX glgA and glgC at OD 0.2
- induction: GlgC at OD 2.3, GlgCAB at OD 2.3, GlgA at OD 1
- use these cultures for glycogen purification via KOH extraction
- End OD values: WT 8.9 CAB 17 C 10.6 A 12,8 deltaX 7.2 deltaP 7.3
10 HPLC samples (of glgB, glgA, WT, ΔglgP, ΔglgX from the 7th September) were given to Philipp: the dried samples were resuspend in 1mL water, centrifuged and filtered. Each in a 1:1 dilution and undiluted.

15-09-11

growth experiment of BL21 glgCAB #1, BL21 WT and BL21 glgC
- iodine staining (OD 1 1 ml) in well plate
- iodine staining with 1 ml OD 5.8
- SDS-Page: from left to right WT, C, CAB (8 ul of OD 12)
glycogen purification via KOH extraction
- use 25 ml LB + 20 mM glucose cultures with BL21 glgCAB #1, WT, ΔglgP, ΔglgX glgA and glgC
- for hydrolysis (for HPLC): do triplicates!

Dinitrosalicylic acid staining (OD of glg A, glg B, glg C, Wildtype, and ΔglgX were adjusted to the OD of ΔglgP = 2.57)
- blank: 1:1 dilution of dinitrosalicylic acid in 0.4 M NaOH

feed glycogen-producing cells to WT
- inoculated 2 BL21 wild type LB precultures (5 ml LB) at 1:20 pm
- inoculate two 10 ml M9 cultures of WT over night
- inoculate one 10 ml LB + 20 mM glucose overnight culture of WT and one of glgCAB
  - induction of glgCAB after 2.5 h
result: no difference in growth could be observed. The hypothesis is: because glycogen degradation enzymes are not secreted, glycogen in the medium does not give cells an advantage in growth when grown on C-limited medium

15-09-12

feed glycogen-producing cells to WT
- inoculated main cultures to an OD of 0.2 at 10:40 am
  - three 25 ml M9 C-limited cultures of WT
  - 3.3 mM glucose, 18.7 mM NH4Cl
- OD at 11:57 am (for calibration): 0.312 (WT1), 0.306 (WT2), 0.308 (WT3)
- measurement was accidently interrupted
- new start OD: 0.357 (WT1), 0.345 (WT2), 0.348 (WT3)
- fed at 5 pm with 500 µl
  - adjust LB WT and the CAB cultures to same OD
  - centrifuge, resolve in 500 µl water and boil for 10 minutes at 95 °C
- WT 1: fed with WT
- WT 2: fed with glgCAB
- WT 3: fed with water

no difference could be observed
the cells do not secrete the glycogen degradation enzymes
boiling might not have disrupted the glycogen chains, therefore, cells might not be able to metabolize the feed
experiment should work if the feed is treated with HCl for acid hydrolysis first

15-09-13

inoculated main cultures to OD 0.182 (ΔglgP + MeOH), 0.18? (#XULU# + ΔglgP + MeOH) and 0.18? (#XULU# + ΔglgP - MeOH)
MeOH (0.3 M) fed at OD 2.8 for ΔglgP
MeOH (0.3 M) fed at OD 2.5 for both #XULU# ΔglgP
in stationary phase, 582 µl MeOH added (finally 0.9 M)

two 10 ml LB overnight cultures of #E4QF# were inoculated
no difference in growth or glycogen production could be observed

15-09-14

End-OD(1)=8,4 End-OD(2)=7,7 End-OD(3)=7,4 (cultures from yesterday)
main cultures inoculated at 9:25 am to OD 0.3 in M9 (C-limited, 3.3 mM Glucose) + K
- cultures 1-3: POLY in ΔglgP + MeOH
- cultures 4-6: POLY in ΔglgP - MeOH

time	OD culture 1	OD 2	OD 3	OD 4	OD 5	OD 6
2	0.341	0.343	0.342	0.335	0.339	0.339
3	0.430	0.425	0.431	0.423	0.431	0.423
4	0.688	0.677	0.698	0.671	0.691	0.649
5	0.802	0.780	0.778	0.775	0.769	0.769
6	0.81	0.83	0.81	0.87	0.78	0.84
7	0.82	0.83	0.85	0.87	0.84	0.95

MeOH added at OD 0.9
- to 22 ml culture volume, 465 µl methanol were added: MeOH concentration of 0.522 M (first thougt the end culture volume would be 23 ml while induction)
- MeOh was added at t: 5 (OD 0.769 - 0.802) at 3:15 p.m.
no influence of MeOH could be observed

15-09-16

cryos of glgCAB in pSB1A30 in ∆glgP (Bl21 Gold DE3) clones #4 (#LOVE#) and #7 (#TEND#)
make LB + 20 mM glucose + IPTG overnight cultures of glgCAB in ∆glgP BL21 Gold (DE3) clone #4 and ∆glgP BL21 Gold (DE3)

15-09-17

adjust glgCAB in ∆glgP BL21 Gold (DE3) clone #4 and ∆glgP BL21 Gold (DE3) to the same OD of 1.97
freeze pellet for iodine staining
*overdays (LB+Antibiotic+IPTG+20 mM glucose) of glgAB in pSB1C30 (#8ZZ4# and #N96D#), glgA in pSB1K30, glgB in pSB1K30 and BL21 WT (incubation since 10 am)
do iodine staining of all cultures in the evening
- glgCAB in ∆glgP BL21 Gold (DE3) clone #4 was stained darker than the ∆glgP BL21 Gold (DE3) culture
- therefore, the combination of the knockoutof glgP and the overexpression of glgCAB leads to higher glycogen production
- for glgAB, no remarkably darker color was observed

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Team:Aachen/Notebook/Documentation/Glycogen Characterization