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
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
| 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
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
- 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#)
- 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