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

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)

• 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

• 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)

• 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

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

• 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 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#)
• 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)

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
• 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