Team:SZ SHD/Lab notes August
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Lab Notes For August
8.1
1. Collecting bacteria through centrifuge
8000rpm, 4℃, 5min
50mL centrifuge tube, dispose supertanant
Resuspend bacteria 5 mL pbs (repeat twice)
Weigh bacteria 3.5g (first collection) 3.8g (second collection)
Add 38mL of pbs and resuspend (0.1g bacteria with 1mL pbs)
Chill and fridge (4℃ for 10min）
sonificate intensity 42% for 30min (work for 6s and rest for 6s)
Centrifuge again 11000rpm for 20min
Collect the supernatant and add equal volume of ELB
Purify protein (check previous procedures on purification)
Ni-Resin tube regeneration
Take 20ul samples and heat for 8min at 95℃: bacterial solution before induction, supernatant after sonification, solution after purification with wash buffer, solution after elusion buffer, and purified R15 protein. (Preparing for gel electrophoresis)
Store protein samples at -20℃
Pictures:
2. Condense
Results:
3. Culture 600 mL of R15 bacteria for 4h at 37℃ and 220 rpm
Check OD and induce with IPTG
8.3
1. Collecting bacteria (what is the name?)
add about 30mL PBS into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
add 5mL PBS into each tube, pippet up and down to resuspend the liquid, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total)
add 5mL PBS into each tube, pippet up and down to resuspend the liquid. put the liquid in all three tubes in one and add PBS until the total volume is about 25mL. Add 10 to 15 uL of isopropanol. Sonificate for 6s and pause for 6s (10min in total, 4 Celsius degree, 42% power) check if more ice is needed. repeat sonification (twice in all)
centrifuge the tube, for 20min,11000rpm, 4 Celsius degree,
remove the supernatant into a new tube. Equal volume of ELB (25mL) is added.
then let ethanol in Ni NTA Beads 6FF Gravity Column off, 5mL (twice) ddH2O passes through to wash the column. 3mL of supernatant-ELB mix passes through, collect the eluate,repeat until all the mix has passes through.
add 5mL (twice) of washing buffer into the column and collect the liquid that passes through with a new tube. 5mL ddH2O passes through to wash. The add 5mL of elation buffer and collect with a new tube, repeat twice (3 times in total)
regenerate the column.
1) Use deionized water (5 column volumes) to wash the column
2) Use 5 column volumes of 100 mM EDTA (pH 8.0) to stripe off nickel ions
3) Use deionized water (10 column volumes) to wash the column
4) Wash with 5 column volumes of 0.5M NaOH (10-15min)
5) Use deionized water (10 column volumes) to wash the column
6) Wash the column with 3-5 column volumes of 100 mM NiSO4 (regenerate hanging Ni)
7) Use deionized water (10 column volumes) to wash the column
Medium to be suspended in 20% ethanol of equal volume at 4 degree celsius if to be saved for future usage
8.4
1. SDS-PAGE
Samples: Z50 EB 1, Z50 EB 2, Z50 EB 3, Z50 WB, Q7 WB.
All sample volumes are 2µL and 2µL of loading buffer is added into each sample.
heat 95℃ 9min
Electrophoresis 120V 200mA 40min
results:
2. Bacterial expansion (in 500mL-flask)
200 mL LB medium, 4% (8mL) bac, 1/1000 (200µL) Ka ✕3
Incubation for 3.5h, 220prm, 37℃
OD value testing
Results:
IPTG induction (1000 µL of IPTG added into each flask)
8.5
1. collecting bacteria Q7 (after 12h of IPTG induction)
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total)
add 5mL PBS into each tube, pippet up and down to resuspend the liquid. put the liquid in all three tubes in one and add PBS until the total volume is about 25mL. Add 15 uL of isopropanol. Sonificate for 6s and pause for 6s (10min in total, 4 Celsius degree, 42% power) check if more ice is needed. repeat sonification (twice in all)
centrifuge the tube, for 20min,11000rpm, 4 Celsius degree, (10 more min of centrifugation because of too much time before supernatant removal.)
weigh the tube, results: supernatant+precipitates 24.78g, precipitates 1.02g, supernatant 23.8g.
remove the supernatant into a new tube. Equal volume of ELB (25mL) is added.
then let ethanol in Ni NTA Beads 6FF Gravity Column off, 5mL (twice) ddH2O passes through to wash the column. 3mL of supernatant-ELB mix passes through, collect the eluate,repeat until all the mix has passes through.
add 5mL (twice) of washing buffer into the column and collect the liquid that passes through with a new tube. 5mL ddH2O passes through to wash. The add 5mL of elation buffer and collect with a new tube, repeat twice (3 times in total)
All the filter is kept in -20℃ fridge
regenerate the column.
[ ] 1) Use deionized water (5 column volumes) to wash the column (actually 5mL is added then dropped, after this another 5mL is passed through, because the liquid flows extremely slow and the first 5mL of ddH2O is used hopefully to remove some impurities.)
[ ] 2) Use 5 column volumes of 100 mM EDTA (pH 8.0) to stripe off nickel ions
[ ] 3) Use deionized water (10 column volumes) to wash the column
[ ] 4) Wash with 5 column volumes of 0.5M NaOH (10-15min) in fact I did 13min
[ ] 5) Use deionized water (10 column volumes) to wash the column
[ ] 6) Wash the column with 3-5 column volumes of 100 mM NiSO4 (regenerate hanging Ni) in fact I did 5 volumes
[ ] 7) Use deionized water (10 column volumes) to wash the column
Medium to be suspended in 20% ethanol of equal volume at 4 degree celsius if to be saved for future usage
2. bacteria expansion
200 mL LB medium, 4% (8mL) bac, 1/1000 (200µL) Ka ✕3
Incubation for about 4h, 220prm, 37℃
OD testing
Results:
IPTG induction (1000 µL of IPTG added into each flask)
3. Do something on the Q7 column
4. make IPTG with concentration of 1M and divide it into several 2mL-tubes
5. Make 2 bottles of 400mL-LB medium and sterilise them in the pot
8.6
1. Collecting bacteria (15h after IPTG induction)
add about 30mL bacteria solution into each of three 50mL tubes. (tube 1,2,3) Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
At the same time, do the same thing as above with tube (4,5,6) in order to improve efficiency
add 5mL PBS into each tube (tube 1,2,3), pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total). And do the same thing with tube 4,5,6
add 3mL PBS into each tube, pippet up and down to resuspend the liquid. put the liquid in all 6 tubes in one and add PBS until the total volume is about 25mL. Add 10 uL of isopropanol. Sonificate for 6s and pause for 6s (10min in total, 4 Celsius degree, 42% power) check if more ice is needed. repeat sonification (twice in all)
centrifuge the tube, for 20min,11000rpm, 4 Celsius degree,
weigh the tube, results: supernatant+precipitates 24.45g, precipitates 1.59g, supernatant 22.87g.
remove the supernatant into a new tube. Keep it in the -20℃ fridge
2. SDS-PAGE
3ul of marker
10ul of empty vector, Q7 (dilute) supertanant, WB (Q7) (dilute), EB (Q7) (eluate1), EB (Q7) (eluate2), EB (Q7) (eluate3), WB (Z50), EB (Z50), EB (Z50) (eluate2), EB (Z50) (eluate3)
8.9
1. Collecting bacteria KU3 (12h of induction under 16℃ and 180rpm)
add about 30mL bacteria solution into each of three 50mL tubes. (tube 1,2,3) Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
At the same time, do the same thing as above with tube (4,5,6) in order to improve efficiency
add 5mL PBS into each tube (tube 1,2,3), pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm,
with proper balance tubes, repeat. (Twice in total). And do the same thing with tube 4,5,6
add 3mL PBS into each tube, pippet up and down to resuspend the liquid. put the liquid in all 6 tubes in one and add PBS until the total volume is about 25mL. Add 10 uL of isopropanol. Sonificate for 6s and pause for 6s (5min in total, 4 Celsius degree, 42% power) check clarity. repeat sonification (four times in all)
centrifuge the tube, for 20min,11000rpm, 4 Celsius degree,
remove the supernatant into a new tube.
weigh the tube, results: precipitates 0.954g, supernatant 24.473g.
Equal volume of ELB (about 25mL) is added into the supernatant.
then let ethanol in Ni NTA Beads 6FF Gravity Column off, 5mL (twice) ddH2O passes through to wash the column. 3mL of supernatant-ELB mix passes through, collect the eluate,repeat until all the mix has passes through.
add 5mL (twice) of washing buffer into the column and collect the liquid that passes through with a new tube. 5mL ddH2O passes through to wash. The add 5mL of elation buffer and collect with a new tube, repeat twice (3 times in total)
All the filter is kept in -20℃ fridge
regenerate the column.
[ ] 1) Use deionized water (5 column volumes) to wash the column (actually 5mL is added then dropped, after this another 5mL is passed through, because the liquid flows extremely slow and the first 5mL of ddH2O is used hopefully to remove some impurities.)
[ ] 2) Use 5 column volumes of 100 mM EDTA (pH 8.0) to stripe off nickel ions
[ ] 3) Use deionized water (10 column volumes) to wash the column
[ ] 4) Wash with 5 column volumes of 0.5M NaOH (10-15min) in fact I did 13min
[ ] 5) Use deionized water (10 column volumes) to wash the column
[ ] 6) Wash the column with 3-5 column volumes of 100 mM NiSO4 (regenerate hanging Ni) in fact I did 5 volumes
[ ] 7) Use deionized water (10 column volumes) to wash the column
Medium to be suspended in 20% ethanol of equal volume at 4 degree celsius if to be saved for future usage
2. Bacteria expansion (Q7)
250 mL LB medium, 3% (7.5mL) bac, 1/1000 (250µL) Ka ✕4
Incubation for about 4h, 220prm, 37℃
OD testing
Results:
IPTG induction (1000 µL of IPTG added into each flask)
3. Bacteria expansion (KU3)
200 mL LB medium, 3% (6mL) bac, 1/1000 (200µL) Ka ✕2
Incubation for about 4h, 220prm, 37℃
OD testing
Results:
IPTG induction (80 µL of IPTG added into each flask)
8.10
1. Collecting bacteria Q7 (12h of induction under 16℃ and 220rpm)
add about 30mL bacteria solution into each of three 50mL tubes. (tube 1,2,3) Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
At the same time, do the same thing as above with tube (4,5,6) in order to improve efficiency
add 5mL PBS into each tube (tube 1,2,3), pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total). And do the same thing with tube 4,5,6
add 3mL PBS into each tube, pippet up and down to resuspend the liquid. put the liquid in all 6 tubes in one and add PBS until the total volume is about 25mL. Add 10 uL of isopropanol. Sonificate for 6s and pause for 6s (25min in total, 4 Celsius degree, 42% power) check clarity with regular time intervals to determine if more time of sonification is needed.
centrifuge the tube, for 20min,11000rpm, 4 Celsius degree,
Remove the supernatant into a new tube.
weigh the tube at the same time, results: supernatant+precipitates: 26.898g
precipitates: 2.934g
2. collecting bacteria KU3 (14h of induction under 16℃ and 220rpm)
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total)
add 5mL PBS into each tube, pippet up and down to resuspend the liquid. put the liquid in all three tubes in one and add PBS until the total volume is about 25mL. Add 10 uL of isopropanol. Sonificate for 6s and pause for 6s (20min in total, 4 Celsius degree, 42% power) check clarity with regular time intervals to determine if more time of sonification is needed.
centrifuge the tube, for 20min,11000rpm, 4 Celsius degree,
Remove the supernatant into a new tube.
weigh the tube at the same time, results: supernatant+precipitates: 24.641g
precipitates: 1.289g
3. Bacteria expansion
200 mL LB medium, 3% (6mL) bac (KU3), 1/1000 (200µL) Ka ✕2
Incubation for about 4h, 220prm, 37℃
200 mL LB medium, 3% (6mL) bac (Q7), 1/1000 (200µL) Ka ✕2
Incubation for about 4h, 220prm, 37℃
200 mL LB medium, 3% (6mL) bac (Z50), 1/1000 (200µL) Ka ✕2
Incubation for about 4h, 220prm, 37℃
OD testing
Results:
IPTG induction (80µL of IPTG added into each flask of KU3, 1000µL of IPTG added into each flask of Q7, 134µL of IPTG added into each flask of Z50)
4. Ultrafiltration of KU3
A. Add 3.5 mL of ddH2O into ultrafiltration tube ; centrifuge for 15min, 4℃, 3500✕g, with similar balance tube. Remove ddH2O
B. Collect all the eluates. Add 3.5 mL of elution buffer; centrifuge for 20min, 3800✕g, 4℃, with similar balance tube. Add some elution buffer into the inner tube until the volume of the elution buffer in the inner tube is about 3.5 mL; pipette up and down to wash the protein stuck on the filter. Repeat centrifugation until the buffer in all three tubes is filtered.
C. Add 200µL of protein preservation solution into ultrafiltration tube and pippet up and down to move and preserve protein in a 2 ml tube
D. Add 3 mL of pbs and centrifuge for 10min, 3800✕g, 4℃, with similar balance tube; repeat. (twice in total)
E. Remove pbs and rinse the ultrafiltration tube with ddH2O. Fill the ultrafiltration tube with 20% ethanol.
5. SDS-PAGE of KU3
Results:
5ul of marker, LANE1: 10ul of empty PET vector, LANE2: KU3 bacterial solution, LANE3: KU3 (dilute) supertanant, LANE4: WB (KU3) (dilute), LANE5: EB (KU3) (eluate1), LANE6: EB (KU3) (eluate2), LANE7: EB (KU3) (eluate3), LANE8: KU3 elute after ultrafiltration
The results showed clear bandings in LANE5 and 8 at ~80kDa, indicating a dimer formed by the target keratinase (KU3). This is not surprising because in some cases the activity of protease attributes to dimer, some do not.
6. Verification of Concentration via BCA kit
Results: the OD (A562) value of KU3 was 0.5566, accordingly the concentration of ultrafitered KU3 was about 0.21 mg/mL.
7. Preliminary tests of ultrafiltered keratinase KU3 on azocasein
Based on the concentration (0.21 mg/mL) tested via OD (A562), the condensed keratinase was diluted into 3 gradients (0.2 mg/mL, 0.1 mg/mL, and 0.04 mg/mL).
120uL keratinase with concentration gradient were added into 480uL of 1% w/v (10mg/mL) azocasein in tris (100mg in 10mL tris), triplicates were done for each gradient.
37°C incubate for 30min and 10h.
Then 0.5N NaOH was added 1:1 to the mixture
OD (A440) was tested by microplate reader.
Results:
8.11
1. Collecting bacteria
A. Z50 (13.5h of induction under 16℃ and 220rpm)
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total)
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria. put the liquid in all three tubes in one and add PBS until the total volume is about 25mL. Add 10 uL of isopropanol. Sonificate for 6s and pause for 6s (20min in total, 4 Celsius degree, 42% power) check clarity with regular time intervals to determine if more time of sonification is needed.
centrifuge the tube, for 25min,11000rpm, 4 Celsius degree,
Remove the supernatant into a new tube.
weigh the tube at the same time, results: supernatant+precipitates: 25.061g
precipitates: 0.957g
B. Q7 (13.5h of induction under 16℃ and 220rpm)
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total)
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria. put the liquid in all three tubes in one and add PBS until the total volume is about 20mL. Add 8 uL of isopropanol. Sonificate for 6s and pause for 6s (11min in total, 4 Celsius degree, 42% power) check clarity with regular time intervals to determine if more time of sonification is needed.
centrifuge the tube, for 25min,11000rpm, 4 Celsius degree,
Remove the supernatant into a new tube.
weigh the tube at the same time, results: supernatant+precipitates: 5.198g
precipitates: 0.492g
PS: during sonification, the sonification stick touched the wall of the tube twice and caused huge amount of foam on the liquid. The foam was removed. That's why the masses of supernatant+precipitates and precipitates are smaller than normal value.
C.
KU3 (15.5h of induction under 16℃ and 220rpm)
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total)
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria. put the liquid in all three tubes in one and add PBS until the total volume is about 25mL. Add 10 uL of isopropanol. Sonificate for 6s and pause for 6s (20min in total, 4 Celsius degree, 42% power) check clarity with regular time intervals to determine if more time of sonification is needed.
centrifuge the tube, for 25min,11000rpm, 4 Celsius degree,
Remove the supernatant into a new tube.
weigh the tube at the same time, results: supernatant+precipitates: 24.323g
precipitates: 1.014g
2. Verification of Concentration via BCA kit after 30min of incubation.
Results:
3. 120uL keratinase crude with concentration gradient were added into 480uL of 1% w/v (10mg/mL) azocasein in tris (100mg in 10mL tris), duplicates were done for each gradient.
4. filter（0.22）to separate, add 1:1 0.5N NaOH
Results of OD (A440):
The result of Z50 indicated its protease activity. While there were too much background noise according to the blank, meaning the protocol should be improved.
Improved:
The blank should contain: 480ul azocasein + 120ul lysis buffer (tris HCl) +1: 1 0.5N NaOH, which means the buffer used before should be also changed to tris, instead of PBS.
8.12
1. Bacterial expansion (KU3, 4h 37℃, 220 rpm)
3% ker KU3 +1L LB broth (250ml in each flask) + 250ul kanamycin for each flask
Incubate at 37°C for about 4hrs to reach an OD (A600) value of 0.5.
OD test:
0.4mM (~100ul) of IPTG added into each flask.
2. After 4 hrs of induction, centrifugation at 10000g, 4°C for 10min. Wheigh the cells.
3. Add 25ml of 50mM tris (pH 7.5).
4. Sonication
5. Centrifugation at 15000g for 20min, then take the supernatant for ultrafiltration
6. Ultrafiltration to get the condensed KU3.
7. Store in tubes
8. Bacterial expansion (KU3) （again, 4h 37℃, 220rpm)
3% ker KU3 +1L LB broth (250ml in each flask) + 250ul kanamycin for each flask
Incubate at 37°C for about 4hrs to reach an OD (A600) value of 0.5.
OD test:
9. IPTG Induction of KU3 (12h, 22℃, 220rpm)
0.4mM (~100ul) of IPTG added into each flask
10. Sonification to obtain crude enzymes
Remove the supernatant into a new tube.
weigh the tube at the same time, results: precipitates 4.592g
11. Concentration of KU3 crude enzyme (determined via BCA kit)
8.13
1. Collecting bacteria
Z50 (induction: 4hrs, 37℃, 220 rpm）
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria, then centrifuge for 1.5min, 8000rpm, with proper balance tubes, repeat. (Twice in total)
add 5mL PBS into each tube, pippet up and down to resuspend the bacteria. put the liquid in all three tubes in one and add PBS until the total volume is about 25mL. Add 10 uL of isopropanol. Sonificate for 6s and pause for 6s (20min in total, 4 Celsius degree, 42% power) check clarity with regular time intervals to determine if more time of sonification is needed.
centrifuge the tube, for 25min,11000rpm, 4 Celsius degree,
Remove the supernatant into a new tube.
weigh the tube at the same time, results: precipitates: 4.881g
2. Keratinase activity assay on KU3
50uL keratinase crude with concentration gradient were added into 200uL of 1% w/v (10mg/mL) azocasein in tris (100mg in 10mL tris).
Incubation under 37℃ for 30min
filter（0.22）to separate, add 1:1 0.5N NaOH
Results of OD (A440):
pH was modified with 0.5N NaOH.
3. Ultrafiltration of KU3.
Add about 3mL of KU3 into the untrafiltration tube and centrifuge for 15min, under 4100xg and 4℃
4. Keratinase activity assay on KU3 (with different concentration of azocacein)
50uL keratinase crude with concentration gradient were added into 200uL of 1% w/v and 3% w/v azocasein in tris buffer.
Incubation under 37℃ for 30min
filter（0.22）to separate, add 1:1 0.5N NaOH
Keratinase activity of KU3
OD value (A440)
5. Concentration of KU3
Group 1: azoc
asein 1%. 0.6172
KU3 1.6048 | 1.0313 | 1.0536 | 1.4302
Blank: 0.6291
Activity:
1) 77
2) 31
3) 26
4) 23
5) 33
6) 108
8.16
1. Bacterial expansion of R15 (4h)
2. [15: 46] IPTG induction of R15
3. Keratinase activity assay of sample 1 (KU3) on 1% azocasein
Concentration gradient: 0.51, 0.25, 0.125 (1, 1/2, 1/4)
The concentration of substrate was too high to get a result within a proper range. Hence the substrate was diluted x10 to reach an OD value (A440) around 0.5,which was consistent with previous value.
8.17
1. Collecting bacteria
R15 (induction: 16hrs, 37℃, 220 rpm）
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
weigh the tube at the same time, precipitates: 5.721g
2. Sonification to obtain crude enzymes
Remove the supernatant into a new tube.
3. Centrigufate to obtain supernatant (crude enzyme)
4. Ultrafitration and diluted the crude enzyme, then divided into EP tubes for activity assays.
5. Re-test of the keratinase activity assay of sample 1 (KU3) on 0.1% azocasein
Concentration gradient: 0.51, 0.25, 0.125 (1, 1/2, 1/4)
1% azocasein was incubated in keratinase solution with the ratio of 4:1, for example 100uL of 1% azocasein was with 25uL of KerBIMKU3. The volume was too small to filter through the 0.22um filter. Consequently there was a loss of liquid after filtration, which might cause an error. We decided to use a larger volume of either the substrate or the keratinase (e.g. 120ul of KU3 and 480ul of azocasein).
7. Modelling of KU3 on azocasein with Michaelis-Menton function.
Protease activity Vmax 78.34248 3.22312
Protease activity Km 0.11507 0.02412
Reduced Chi-sqr = 10.809500293
COD(R^2) = 0.97349493869704
Iterations Performed = 8
Total Iterations in Session = 8
Fit converged. Chi-Sqr tolerance value of 1E-9 was reached.
Protease activity 78.34248 3.22312 0.11507 0.02412 10.8095 0.96024
8.18
1. [11:30] Bacterial expansion (Z50, 4h 37℃, 220 rpm)
3% ker Z50 +1L LB broth (250ml in each flask) + 250ul kanamycin for each flask
Incubate at 37°C for about 4hrs to reach an OD (A600) value of 0.5.
OD test:
2. [16:00] IPTG Induction of Z50 ( 16h, 28℃, 220 rpm)
0.4mM (~150ul) of IPTG added into each flask.
3. Transform the stock of Z50 into a 50ml tube.
4. Concentration of KU3, Z50, and R15
Standard curve:
The BCA results showed even 16 times of dilution was too condensed to be detected within the proper range of OD value, which would be inaccurate.
5. Keratinase activity assay of KU3, Z50 and R15 on 0.1% azocasein (0.1g/100ml)
Prepare a gradient of keratinase (1, 1/2, 1/4, 1/8, 1/16)
8.19
1. Collecting bacteria
Z50 (induction: 16hrs, 28℃, 220 rpm）
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
weigh the tube at the same time, precipitates: 5.321g
3. Sonification to obtain crude enzymes
Remove the supernatant into a new tube.
4. Centrigufate to obtain supernatant (crude enzyme)
5. Ultrafitration and diluted the crude enzyme, then divided into EP tubes for activity assays.
6. Take one tube of 1ml keratinse for activity assay, others are kept in the -80°C fridge.
7. To analyze, the concentration of 1/200 dilute fell in the range of standard concentration determined by BCA kit. According to OD (A440), the concentration of 1/200 dilute was around 0.27 mg/mL. In reference to this value, the concentration of original Z50 protein solution was around 54mg/mL.
8. [11:30] Bacterial expansion of Q7 (4h 37℃, 220 rpm)
3% kerQ7 +1L LB broth (250ml in each flask) + 250ul kanamycin for each flask
Incubate at 37°C for about 4hrs to reach an OD (A600) value of 0.5.
OD test:
9. [16:30] IPTG Induction of Q7 ( 16h, 28℃, 220 rpm)
~1250ul of IPTG added into each flask.
8.20
1. Collecting bacteria
Q7 (induction: 16hrs, 28℃, 220 rpm）
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
remove the supernatant.
Repeat until all the liquid has been centrifuged.
weigh the tube at the same time, precipitates: 5.211g
3. Sonification to obtain crude enzymes. No isobutanol was added, and there's no bubbles.
Remove the supernatant into a new tube.
4. Centrigufate to obtain supernatant (crude enzyme)
5. Ultrafitration and diluted the crude enzyme, then divided into EP tubes for activity assays.
8.22
OD:
1. IPTG Induction of Q7 ( 16h, 16℃, 220 rpm)
~1250ul of IPTG added into each flask.
8.23
1. Collecting bacteria
Q7 (induction: 16hrs, 16℃, 220 rpm）
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
weigh the tube at the same time, precipitates: 5.581g
2. Sonification to obtain crude enzymes. No isobutanol was added, and there's no bubbles.
Remove the supernatant into a new tube.
3. Keratinase activity assay
Substrates: azocaein, casein, gelatin, BSA, hair
- Azocasein (triplicates):
120 µL of an appropriate dilution of enzyme (ultrafiltrated crude enzyme/dilutes) was added to 480 µL of azocasein (1% w/v) in Tris buffer (50 mmol/L, pH 7.5) and the mixture was incubated at 37°C for 30 min. The reaction was terminated by filtering through a 0.22um filter. 100ml of the supernatant was neutralized (~pH 7.5)by adding 100ml of 0.5N NaOH. Absorbance (A440) was measured using a spectrophotometer.
One unit of protease activity was defined as the amount of enzyme required to yield an increase in absorbance (A420) of 0.01 in 30 min at 37°C.
- Casein (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme/dilutes) solution (0.5 ml) was mixed with 2.5 ml 100 mM Glycine-NaOH buffer supplemented with 2 mM CaCl2 containing 1% w/v (500mg/50ml) casein (prepare 50ml stock), and incubated for 15 min at suitable pH and temperature (pH7.5, 37°C). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control. One unit (U) of caseinolytic activity was defined as the amount of enzyme that hydrolyzed the substrate and produced 1 mg of amino acid equivalent to tyrosine per minute under the above-mentioned conditions.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
Prepare 50ml 2 mM CaCl2 stock solution (M) by adding: Dissolve 0.0147g of CaCl2.2H2O
Prepare 500 mM Na2CO3: 2655 mg Na2CO3
Add distilled water until volume is 50 mL.
- Gelatin (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme)solution (0.5 ml) was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) gelatin (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
- BSA (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme)solution (0.5 ml) was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) BSA (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
4. Quantification of Sulfhydryl Groups in hydrolysate
Catalytic Mechanism
The mechanism by which microorganisms degrade keratin varies, so the product during degradation is not the same. Some fungi reduce the disulfide bonds through the sulfites secreted on the surface of the mycelia and the acidic environment, while Streptomyces through the production of intracellular reductase. However, water-insoluble keratin can only exist extracellularly in the form of particles. Therefore, the reduction of disulfide bonds can only occur outside the whole cell with strong metabolic ability, most likely in the cell-bound redox system on the cell surface because it requires insoluble keratin in close contact with cells. Observations of pure white high-temperature actinomycetes revealed that the disulfide bond reduction was performed by a cell-linked redox system. No sulfhydryl groups were detected during keratolysis of S.
freundii and B. licheniformis. This may be due to the fact that the cysteine (-SH) produced by the reduction of the cystine disulfide bond was quickly converted to other product.
Keratinase actually has the activity of polypeptide hydrolase and disulfide reductase. At present, it is generally believed that the degradation process of keratinase is divided into three steps, namely denaturation, hydrolysis and transamination. First, the disulfide reductase acts on the keratin disulfide bond to reduce cystine (-S-S-) to cysteine (-SH), so that the high-level structure of keratin disintegrates to form degenerative keratin protein. The degenerative keratin protein is gradually hydrolyzed into polypeptides, oligopeptides and free amino acids by the action of polypeptide hydrolase. Finally, ammonia and sulfide are produced by transamination to completely hydrolyze keratin.
Procedure for Quantitating Sulfhydryl Groups Based on Molar Absorptivity
A. Material Preparation
• Reaction Buffer: 0.1M sodium phosphate, pH 8.0, containing 1mM EDTA (50ml stock)
• Ellman’s Reagent Solution: Dissolve 4mg Ellman’s Reagent in 1mL of Reaction Buffer.
B. Measure Absorbance
1. For each unknown sample to be tested, prepare a tube containing 50μL of Ellman’s Reagent Solution and 2.5mL of Reaction Buffer.
2. Add 250μL of each unknown to the separate test tubes prepared in step 1. As a blank, add 250μL of Reaction Buffer to a separate test tube prepared in Step 1.
Note: For the unknown(s), make dilutions so that the 250μL sample applied to the assay reaction has a sulfhydryl concentration less than 1.0mM. Concentrations exceeding 1mM free sulfhydryl will result in high absorbance values and less accurate estimation of the concentration based on the extinction coefficient.
3. Mix and incubate at room temperature for 15 min.
4. With a spectrophotometer set to 412nm, zero the instrument on the blank and then measure absorbance of each sample.
5. Calculate the amount and concentration of sulfhydryls in the sample from the molar extinction coefficient of TNB.
8.25
1. Sonification of Q7 to obtain crude enzymes. No isobutanol was added, and there's no bubbles.
Remove the supernatant into a new tube.
2. Ultrafitration of crude enzymes, dilute to testify its concentration.
3. Concentration gradient of keratinase activity on 0.1% azocacein, following Aug 18th.
4. Supplement of BCA test on Aug 18th.
Since 100 times dilute was not within the curve, the data was not used to calculate the original concentration. According to the concentration of 200 times dilutes, the original concentrations were calculated as 67.53mg/ml (KU3), 49.07mg/ml (Z50), 69.4mg/ml (R15), and 55.05mg/ml (Q7).
5. [11:00] Bacterial cultivation and IPTG induction of R15 (1L): 100ul IPTG, 37°C 16h.
6. SDS-PAGE of crude enzymes
Figure. SDS-PAGE stained with Fast Protein Stain. Protein molecular marker; Lane1. 10 times dilute of crude enzyme (kerBIMKU3); Lane2. 100 times dilute of crude enzyme (kerBIMKU3); Lane3. kerBIMKU3 bacterial solution before induction. Lane4. 10 times dilute of crude enzyme (kerAvDZ50); Lane5. 100 times dilute of crude enzyme (kerAvDZ50); Lane6. kerAvDZ50 bacterial solution before induction; Lane7. 10 times dilute of crude enzyme (kerBlER15); lane8. 100 times dilute of crude enzyme (kerBlER15); Lane9. kerBlER15 bacterial solution before induction; Lane10. 10 times dilute of crude enzyme (kerBteQ7); Lane11. 100 times dilute of crude enzyme (kerBteQ7); Lane12. kerBteQ7 bacterial solution before induction;
8.26
1. Collecting bacteria
R15 (induction: 16hrs, 16℃, 220 rpm）
add about 30mL bacteria solution into each of three 50mL tubes. Centrifuge for 5min, 8000rpm, 4℃, with proper balance tubes.
weigh the tube at the same time, precipitates: 7.015g
8.27
Activity assay of R15
- [14:50] Casein (triplicates): pH9 forgot to adjust the pH
A suitably diluted enzyme (69.4 mg/ml, 34.7 mg/ml, 0.694 mg/ml, 0.347 mg/ml respectively, ultrafiltrated crude enzyme/dilutes) solution (0.5 ml) was mixed with 2.5 ml 100 mM Glycine-NaOH buffer supplemented with 2 mM CaCl2 containing 1% w/v (500mg/50ml) casein (prepare 50ml stock), and incubated for 15 min at suitable pH and temperature (pH7.5, 37°C). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control. One unit (U) of caseinolytic activity was defined as the amount of enzyme that hydrolyzed the substrate and produced 1 mg of amino acid equivalent to tyrosine per minute under the above-mentioned conditions.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
- BSA (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme)solution (0.5 ml) was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) BSA (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
8.30
OD:
1. [15:00] IPTG Induction of KU3 ( 37h, 4℃, 220 rpm)
~120ul of IPTG added into each flask.
2. Collecting bacteria
8.31
1. Activity assay for Q7
- [1:30] Casein (triplicates): adjust pH7.5
A suitably diluted enzyme (55.05 mg/ml, 27.525 mg/ml, 5.505 mg/ml, 27.525 mg/ml respectively, ultrafiltrated crude enzyme/dilutes) solution (0.5 ml) was mixed with 2.5 ml 100 mM Glycine-NaOH buffer supplemented with 2 mM CaCl2 containing 1% w/v (500mg/50ml) casein (prepare 50ml stock), and incubated for 15 min at suitable pH and temperature (pH7.5, 37°C). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control. One unit (U) of caseinolytic activity was defined as the amount of enzyme that hydrolyzed the substrate and produced 1 mg of amino acid equivalent to tyrosine per minute under the above-mentioned conditions.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
30min & 12hrs
R15 was not tested, due to its lower expression compared with the other keratinases.
Results: It's hard to tell the difference between each enzyme, since the concentration of substrate (casein) is too high.
- [2:00] Gelatin (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme)solution (0.5 ml) was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) BSA (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
30min & 12hrs
Results: KU3 showed better enzyme activity in gelatin compared with the other two enzymes. Compared to casein, the effect of enzymes is lower.