Team:SZ SHD/Lab Notes September
Lab Notes for September
1st of September
1. Soncation and Ultrafiltration of KU3, keep in -80°C.
2. Plate streaking of 4 bacteria
3. Dilute casein to 0.1% w/v and test for enzyme activity again.
| Blank | Control | KU3 | Z50 | Q7 | R15 |
|---|---|---|---|---|---|
| 500ul of 100mM Glycine-NaOH buffer 2.5ml Na2CO3 500ul Folin reagent |
500ul of 100mM Glycine-NaOH buffer with 0.1% cacein 2.5ml Na2CO3 500ul Folin reagent |
Ori 1/2 1/4 1/10 1/100 |
Ori 1/2 1/4 1/10 1/100 |
Ori 1/2 1/4 1/10 1/100 |
Ori 1/2 1/4 1/10 1/100 |
30min & 12hrs
Fig. Protease activity of 4 keratinases on 0.1% casein at 30 min of incubation, pH 7.5.
Calculate concentration - see Sep 2nd
Fig. Protease activity of 4 keratinases on 1% gelatin at 30 min of incubation, pH 7.5.
2nd of September
1. BCA test to determine concentration.
| Enzyme concetration (mg/ml) | Correction coefficient (to 100 mg/ml) | |
|---|---|---|
| 400 times dilute of kU3 | 0.35 | |
| 400 times dilute of Z50 | 0.35 | |
| 400 times dilute of Q7 | 0.27 | |
| 400 times dilute of R15 | 0.48 | |
| KU3 ori | 139.09 | 1.39 |
| Z50 ori | 139.73 | 1.4 |
| Q7 ori | 108 | 1.08 |
| R15 ori | 191.55 | 1.92 |
2. Protease activity of 4 keratinases on 0.1% BSA.
| Blank | Control | KU3 | Z50 | Q7 | R15 |
|---|---|---|---|---|---|
| 500ul of 100mM Glycine-NaOH buffer 2.5ml Na2CO3 500ul Folin reagent |
500ul of 100mM Glycine-NaOH buffer with 0.1% BSA 2.5ml Na2CO3 500ul Folin reagent |
Ori 1/2 1/4 1/10 1/100 |
Ori 1/2 1/4 1/10 1/100 |
Ori 1/2 1/4 1/10 1/100 |
Ori 1/2 1/4 1/10 1/100 |
30min & 12hrs
Fig. Protease activity of 4 keratinases on 0.1% BSA at 30 min of incubation, pH 7.5.
Fig. Protease activity of 4 keratinases on different natural substrates at 30 min of incubation, pH 7.5. The degree of action of keratinase on the substrate is in the order of BSA, casein, and gelatin. KU3, Z50 and Q7 all showed a relatively high enzyme activity among 4 keratinases at a higher concentration (100mg/ml) on the three substrates (casein, BSA and gelatin). Whereas, the activity of Z50 is relatively lower at a lower concentration (<50 mg/ml), compared with KU3 and Q7.
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.
Note: according to previous record and results, R15 should not be our focus.
3rd of September
1. DTNB pre-test
Reagent:
Dissolve 39.6 mg. of 5,5’-dithiobis(2nitrobenzoie acid) (DTNB) in 10 ml phosphate (p = 0.1) buffer (pH 7.0).
Weighed the hair sample
Treat the hair sample with keratinase for 30 min., and there should be free thiol groups in the solution.
Filter the treated solution.
Solution 3 ml (if the volume is lower than 3 ml, dilute then calculate the original)
Buffer (tris, pH 8.0) 2 ml
H2O 5 ml
The reagent (0.02 ml.) was added to 3.0 ml. of this mixture in a photometer cell.
The color developed rapidly (2 min.), and the absorbance at 412 nm was tested.
| Blank | Control | Treated Sample (15 min) | Treated Sample (15 min) | Treated Sample (15 min) | |
|---|---|---|---|---|---|
| DTNB reagent pH7 | Substrate + DTNB | Keratinase KU3 + substrate + DTNB |
Keratinase Q7+ substrate + DTNB |
20mg/ml DTT + substrate + DTNB | |
| OD (A412) after 15min of incubation | 0.5337 | 0.5391 | 0.6314 | 0.9116 | 1.3275 |
C0=(A/ε)*D
A= abs at 412 mu
ε=13600/M/cm (extinction coefficient)
D=dilution factor
Eg. C0=(A/13600)*(10/x)*[(x+0.02)/x]
6th of September
1. Q7 R15 monoclonal clones were selected from the plate and inoculated into 6ml antibiotic containing medium overnight at 200rpm at 37℃
7th of of September
1. [12:00] Overnight cultures were inoculated with 1% into 600ml LB containing antibiotics, and incubated at 37 ° C at 200rpm in violent shaking for about 4h
150mL for each flask, since LB broth is not enough, 1 exception with 100mL for Q7.
2. [16:20] IPTG Induction of R15, Q7 ( 15h, 16℃, 180 rpm)
~63 uL of IPTG added into each R15 flask.
~750uL of IPTG added into flasks with 150mL Q7
~500uL of IPTG added into a flask with 100mL Q7
8th of September
1. Keratinase activity assay of KU3 on azocasein
- Azocasein (triplicates):
- In Azocasein method you measure any free aminoácid or peptide containing the Azo dye.
100 µL of an appropriate dilution of enzyme (ultrafiltrated crude enzyme/dilutes) was added to 400 µL of azocasein (0.05，0.1，0.5，1% w/v) in tris buffer (50 mmol/L, pH 7.5) and the mixture was incubated at 37°C. The reaction was terminated by filtering through a 0.22um filter. 100ul of the supernatant was neutralized (~pH 7.5) by adding 100ul of 0.5N NaOH.
Absorbance (A440) was measured using a spectrophotometer.
- BCA test of KU3
The original concentration of KU3 was 94.5mg/ml. The correction coefficient to 100 was 0.945.
| KU3 activity | 0min | 5min | 10min | 15min | 20min | 25min | 30min | 12h | 24h | 36h | ||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| azocasein %w/v | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv |
| 1 | 111.746 | 142.328 | 156.9312 | 137.0018 | 23.05867 | 299.7884 | 300 | 326.2434 | 308.6772 | 15.21309 | 335.6614 | 335.4497 | 340.2116 | 337.1076 | 2.690274 | 344.5503 | 345.3968 | 346.455 | 345.4674 | 0.954339 | 364.2328 | 358.2011 | 359.4709 | 360.6349 | 3.17989 | 364.127 | 364.6561 | 364.0212 | 364.2681 | 0.340164 | 381.2698 | 368.6772 | 366.5608 | 372.1693 | 7.952013 | 390.582 | 384.5503 | 391.3228 | 388.8183 | 3.714773 | 361.4815 | 390.582 | 379.2593 | 377.1076 | 14.6691 | 346.5608 | 361.4815 | 373.1217 | 360.388 | 13.31414 |
| 0.5 | 103.4921 | 100 | 97.98942 | 100.4938 | 2.784363 | 161.164 | 159.7884 | 130.1587 | 150.3704 | 17.5173 | 165.9259 | 160.1058 | 163.8095 | 163.2804 | 2.945907 | 169.8413 | 164.9735 | 171.2169 | 168.6772 | 3.280423 | 166.8783 | 176.4021 | 174.1799 | 172.4868 | 4.982543 | 173.4392 | 178.4127 | 174.2857 | 175.3792 | 2.660978 | 192.381 | 185.6085 | 186.1376 | 188.0423 | 3.766661 | 251.5344 | 239.0476 | 241.2698 | 243.9506 | 6.661065 | 251.5344 | 239.0476 | 241.2698 | 243.9506 | 6.661065 | 229.418 | 219.8942 | 243.5979 | 230.97 | 11.92782 |
| 0.1 | 3.915344 | 3.354497 | 3.703704 | 3.657848 | 0.283221 | 44.98413 | 45.5873 | 44.85714 | 45.14286 | 0.390102 | 47.0582 | 46.10582 | 45.62963 | 46.26455 | 0.727393 | 48.43386 | 46.9418 | 48.81481 | 48.06349 | 0.989913 | 52.09524 | 51.48148 | 49.52381 | 51.03351 | 1.342971 | 54.70899 | 55.21693 | 55.04762 | 54.99118 | 0.258629 | 56.16931 | 57.3545 | 56.22222 | 56.58201 | 0.669516 | 140.3175 | 138.0952 | 147.619 | 142.0106 | 4.982543 | 133.2275 | 132.9101 | 129.2063 | 131.7813 | 2.235619 | 113.4392 | 94.01058 | 123.3862 | 110.2787 | 14.94068 |
| 0.05 | 21.48148 | 31.53439 | 22.1164 | 25.04409 | 5.629722 | 24.83598 | 25.12169 | 24.91005 | 24.95591 | 0.148274 | 26.40212 | 25.32275 | 25.67196 | 25.79894 | 0.550773 | 26.08466 | 26.97354 | 26.93122 | 26.66314 | 0.501428 | 28.28571 | 28.32804 | 29.40741 | 28.67372 | 0.635743 | 32.50794 | 30.15873 | 31.33333 | 31.33333 | 1.174603 | 36.25397 | 32.38095 | 35.01587 | 34.55026 | 1.978044 | 125.1852 | 122.2222 | 130.2646 | 125.8907 | 4.067312 | 115.4497 | 114.6032 | 107.3016 | 112.4515 | 4.479996 | 106.7725 | 104.6878 | 105.6402 | 105.7002 | 1.043621 |
2. Collecting bacteria Q7, R15
Q7 be centrifuged, and PBS washed
3. Culture KU3 5ml overnight (37°C, 180rpm)
4. Culture KU3, Q7, R15, DZ50 5ml overnight (37°C, 180rpm) for glycerol preservation
9th of September
1. 16:00 Incubating bacteria: KU3 1%, 100ml*6 overnight (180rpm, 37°C)
2. sonication
10th of September
1. Keratinase activity assay of Q7 on azocasein
- Azocasein (triplicates):
- In Azocasein method you measure any free aminoácid or peptide containing the Azo dye.
100 µL of an appropriate dilution of enzyme (ultrafiltrated crude enzyme/dilutes) was added to 400 µL of azocasein (0.05，0.1，0.5，1% w/v) in tris buffer (50 mmol/L, pH 7.5) and the mixture was incubated at 37°C. The reaction was terminated by filtering through a 0.22um filter. 100ul of the supernatant was neutralized (~pH 7.5) by adding 100ul of 0.5N NaOH.
Absorbance (A440) was measured using a spectrophotometer.
- BCA test of Q7
| Standard concetration (mg/ml) | 0 | 0.025 | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 | 0.458 |
|---|---|---|---|---|---|---|---|---|---|
| OD (A562) | 0.122 | 0.1352 | 0.1451 | 0.1557 | 0.1851 | 0.205 | 0.2348 | 0.2542 | 0.2472 |
The original concentration of KU3 was 91.61mg/ml. The correction coefficient to 100 was 0.9161.
14th of September
4:40 culture Q7, KU3 each 6ml (37℃, 180rpm) overnight
1. collecting bacteria KU3, centrifuged 4℃, PBS washed 3 times. (-20℃ stored overnight.)
15th of September
1. 7:30 expansion of KU3, 1%, 600ml (37℃, 180rpm)
2. Lunch time IPTG expression, 40 ul to every 100 ml KU3 and 500ul to every 100 ml Q7, 600 ml in total for each keratinase
3. 4:00 ultrasonication, (centrifuge 4℃, 8000rpm, 10min) crude enzyme stored in -20℃ overnight
4. collecting bacteria-
17th of September
response surface - protease activity
| KU3 OD | 0min | 5min | 10min | 15min | 20min | 25min | 30min | 12h | 24h | 36h | ||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| azocasein %w/v | sample 1 | sample 2 | sample 3 | average | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv |
| 1 | 1.056 | 1.345 | 1.483 | 1.294667 | 0.217904 | 2.833 | 2.835 | 3.083 | 2.917 | 0.143764 | 3.172 | 3.17 | 3.215 | 3.185667 | 0.025423 | 3.256 | 3.264 | 3.274 | 3.264667 | 0.009018 | 3.442 | 3.385 | 3.397 | 3.408 | 0.03005 | 3.441 | 3.446 | 3.44 | 3.442333 | 0.003215 | 3.603 | 3.484 | 3.464 | 3.517 | 0.075147 | 3.691 | 3.634 | 3.698 | 3.674333 | 0.035105 | 3.416 | 3.691 | 3.584 | 3.563667 | 0.138623 | 3.275 | 3.416 | 3.526 | 3.405667 | 0.125819 |
| 0.5 | 0.978 | 0.945 | 0.926 | 0.949667 | 0.026312 | 1.523 | 1.51 | 1.23 | 1.421 | 0.165539 | 1.568 | 1.513 | 1.548 | 1.543 | 0.027839 | 1.605 | 1.559 | 1.618 | 1.594 | 0.031 | 1.577 | 1.667 | 1.646 | 1.63 | 0.047085 | 1.639 | 1.686 | 1.647 | 1.657333 | 0.025146 | 1.818 | 1.754 | 1.759 | 1.777 | 0.035595 | 2.377 | 2.259 | 2.28 | 2.305333 | 0.062947 | 2.377 | 2.259 | 2.28 | 2.305333 | 0.062947 | 2.168 | 2.078 | 2.302 | 2.182667 | 0.112718 |
| 0.1 | 0.037 | 0.0317 | 0.035 | 0.034567 | 0.002676 | 0.4251 | 0.4308 | 0.4239 | 0.4266 | 0.003686 | 0.4447 | 0.4357 | 0.4312 | 0.4372 | 0.006874 | 0.4577 | 0.4436 | 0.4613 | 0.4542 | 0.009355 | 0.4923 | 0.4865 | 0.468 | 0.482267 | 0.012691 | 0.517 | 0.5218 | 0.5202 | 0.519667 | 0.002444 | 0.5308 | 0.542 | 0.5313 | 0.5347 | 0.006327 | 1.326 | 1.305 | 1.395 | 1.342 | 0.047085 | 1.259 | 1.256 | 1.221 | 1.245333 | 0.021127 | 1.072 | 0.8884 | 1.166 | 1.042133 | 0.141189 |
| 0.05 | 0.203 | 0.298 | 0.209 | 0.236667 | 0.053201 | 0.2347 | 0.2374 | 0.2354 | 0.235833 | 0.001401 | 0.2495 | 0.2393 | 0.2426 | 0.2438 | 0.005205 | 0.2465 | 0.2549 | 0.2545 | 0.251967 | 0.004738 | 0.2673 | 0.2677 | 0.2779 | 0.270967 | 0.006008 | 0.3072 | 0.285 | 0.2961 | 0.2961 | 0.0111 | 0.3426 | 0.306 | 0.3309 | 0.3265 | 0.018693 | 1.183 | 1.155 | 1.231 | 1.189667 | 0.038436 | 1.091 | 1.083 | 1.014 | 1.062667 | 0.042336 | 1.009 | 0.9893 | 0.9983 | 0.998867 | 0.009862 |
| KU3 activity | 0min | 5min | 10min | 15min | 20min | 25min | 30min | 12h | 24h | 36h | ||||||||||||||||||||||||||||||||||||||||
| azocasein %w/v | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv |
| 1 | 111.746 | 142.328 | 156.9312 | 137.0018 | 23.05867 | 299.7884 | 300 | 326.2434 | 308.6772 | 15.21309 | 335.6614 | 335.4497 | 340.2116 | 337.1076 | 2.690274 | 344.5503 | 345.3968 | 346.455 | 345.4674 | 0.954339 | 364.2328 | 358.2011 | 359.4709 | 360.6349 | 3.17989 | 364.127 | 364.6561 | 364.0212 | 364.2681 | 0.340164 | 381.2698 | 368.6772 | 366.5608 | 372.1693 | 7.952013 | 390.582 | 384.5503 | 391.3228 | 388.8183 | 3.714773 | 361.4815 | 390.582 | 379.2593 | 377.1076 | 14.6691 | 346.5608 | 361.4815 | 373.1217 | 360.388 | 13.31414 |
| 0.5 | 103.4921 | 100 | 97.98942 | 100.4938 | 2.784363 | 161.164 | 159.7884 | 130.1587 | 150.3704 | 17.5173 | 165.9259 | 160.1058 | 163.8095 | 163.2804 | 2.945907 | 169.8413 | 164.9735 | 171.2169 | 168.6772 | 3.280423 | 166.8783 | 176.4021 | 174.1799 | 172.4868 | 4.982543 | 173.4392 | 178.4127 | 174.2857 | 175.3792 | 2.660978 | 192.381 | 185.6085 | 186.1376 | 188.0423 | 3.766661 | 251.5344 | 239.0476 | 241.2698 | 243.9506 | 6.661065 | 251.5344 | 239.0476 | 241.2698 | 243.9506 | 6.661065 | 229.418 | 219.8942 | 243.5979 | 230.97 | 11.92782 |
| 0.1 | 3.915344 | 3.354497 | 3.703704 | 3.657848 | 0.283221 | 44.98413 | 45.5873 | 44.85714 | 45.14286 | 0.390102 | 47.0582 | 46.10582 | 45.62963 | 46.26455 | 0.727393 | 48.43386 | 46.9418 | 48.81481 | 48.06349 | 0.989913 | 52.09524 | 51.48148 | 49.52381 | 51.03351 | 1.342971 | 54.70899 | 55.21693 | 55.04762 | 54.99118 | 0.258629 | 56.16931 | 57.3545 | 56.22222 | 56.58201 | 0.669516 | 140.3175 | 138.0952 | 147.619 | 142.0106 | 4.982543 | 133.2275 | 132.9101 | 129.2063 | 131.7813 | 2.235619 | 113.4392 | 94.01058 | 123.3862 | 110.2787 | 14.94068 |
| 0.05 | 21.48148 | 31.53439 | 22.1164 | 25.04409 | 5.629722 | 24.83598 | 25.12169 | 24.91005 | 24.95591 | 0.148274 | 26.40212 | 25.32275 | 25.67196 | 25.79894 | 0.550773 | 26.08466 | 26.97354 | 26.93122 | 26.66314 | 0.501428 | 28.28571 | 28.32804 | 29.40741 | 28.67372 | 0.635743 | 32.50794 | 30.15873 | 31.33333 | 31.33333 | 1.174603 | 36.25397 | 32.38095 | 35.01587 | 34.55026 | 1.978044 | 125.1852 | 122.2222 | 130.2646 | 125.8907 | 4.067312 | 115.4497 | 114.6032 | 107.3016 | 112.4515 | 4.479996 | 106.7725 | 104.6878 | 105.6402 | 105.7002 | 1.043621 |
| Q7 OD | 0min | 5min | 10min | 15min | 20min | 25min | 30min | 12h | 24h | 36h | ||||||||||||||||||||||||||||||||||||||||
| azocasein %w/v | sample 1 | sample 2 | sample 3 | average | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv |
| 1 | 0.9111 | 0.8941 | 0.9449 | 0.9167 | 0.025859 | 1.917 | 0.9358 | 1.928 | 1.5936 | 0.569698 | 1.972 | 0.9526 | 1.9586 | 1.627733 | 0.584721 | 2.355 | 2.432 | 2.474 | 2.420333 | 0.060352 | 1.9701 | 3.037 | 3.093 | 2.700033 | 0.632761 | 3.249 | 3.174 | 2.334 | 2.919 | 0.508011 | 3.0789 | 3.093 | 3.249 | 3.1403 | 0.094401 | 3.591 | 3.714 | 3.839 | 3.714667 | 0.124001 | 2.866 | 3.2068 | 3.3278 | 3.133533 | 0.239459 | 2.9692 | 0.9526 | 1.9701 | 1.963967 | 1.008314 |
| 0.5 | 0.5847 | 0.5689 | 0.6108 | 0.588133 | 0.02116 | 0.5865 | 0.6106 | 0.6123 | 0.603133 | 0.01443 | 0.6363 | 0.6263 | 0.6283 | 0.6303 | 0.005292 | 0.6539 | 1.088 | 1.12 | 0.953967 | 0.260357 | 1.085 | 1.056 | 1.141 | 1.094 | 0.043209 | 1.65 | 1.166 | 1.726 | 1.514 | 0.303763 | 1.7538 | 1.6116 | 1.7121 | 1.6925 | 0.073098 | 2.377 | 2.302 | 1.995 | 2.224667 | 0.202401 | 0.9471 | 0.9812 | 1.441 | 1.1231 | 0.275837 | 0.7342 | 0.564 | 0.8649 | 0.721033 | 0.150881 |
| 0.1 | 0.1803 | 0.1763 | 0.1848 | 0.180467 | 0.004252 | 0.2069 | 0.218 | 0.2198 | 0.2149 | 0.006986 | 0.2245 | 0.2219 | 0.2486 | 0.231667 | 0.014722 | 0.2614 | 0.4494 | 0.4576 | 0.389467 | 0.110985 | 0.4517 | 0.4472 | 0.455 | 0.4513 | 0.003915 | 0.4768 | 0.4599 | 0.4835 | 0.4734 | 0.012162 | 0.5328 | 0.5163 | 0.5416 | 0.530233 | 0.012844 | 1.248 | 1.0844 | 1.327 | 1.2198 | 0.123734 | 0.8825 | 0.8537 | 0.8834 | 0.8732 | 0.016893 | 0.7835 | 0.5527 | 0.8537 | 0.729967 | 0.157479 |
| 0.05 | 0.1617 | 0.1578 | 0.1664 | 0.161967 | 0.004306 | 0.1879 | 0.1995 | 0.1909 | 0.192767 | 0.006021 | 0.1947 | 0.2033 | 0.2296 | 0.2092 | 0.018183 | 0.2325 | 0.3428 | 0.3451 | 0.3068 | 0.064356 | 0.3491 | 0.3471 | 0.3726 | 0.356267 | 0.01418 | 0.3703 | 0.3737 | 0.3743 | 0.372767 | 0.002157 | 0.4325 | 0.4399 | 0.4579 | 0.443433 | 0.013063 | 1.1834 | 1.1099 | 1.0182 | 1.103833 | 0.082767 | 0.8634 | 0.8555 | 0.8176 | 0.8455 | 0.024483 | 0.809 | 0.5545 | 0.8555 | 0.739667 | 0.162036 |
| Q7 activity | 0min | 5min | 10min | 15min | 20min | 25min | 30min | 12h | 24h | 36h | ||||||||||||||||||||||||||||||||||||||||
| azocasein %w/v | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv | sample 1 | sample 2 | sample 3 | ave | sdv |
| 1 | 99.46507 | 97.60917 | 103.155 | 100.0764 | 2.823018 | 209.2795 | 102.1616 | 210.4803 | 173.9738 | 62.19411 | 215.2838 | 103.9956 | 213.821 | 177.7001 | 63.83417 | 257.0961 | 265.5022 | 270.0873 | 264.2285 | 6.588619 | 215.0764 | 331.5502 | 337.6638 | 294.7635 | 69.07867 | 354.6943 | 346.5066 | 254.8035 | 318.6681 | 55.4597 | 336.1245 | 337.6638 | 354.6943 | 342.8275 | 10.30574 | 392.0306 | 405.4585 | 419.1048 | 405.5313 | 13.53726 | 312.8821 | 350.0873 | 363.2969 | 342.0888 | 26.14186 | 324.1485 | 103.9956 | 215.0764 | 214.4068 | 110.0779 |
| 0.5 | 63.83188 | 62.10699 | 66.68122 | 64.2067 | 2.310038 | 64.02838 | 66.65939 | 66.84498 | 65.84425 | 1.575322 | 69.46507 | 68.37336 | 68.5917 | 68.81004 | 0.577675 | 71.38646 | 118.7773 | 122.2707 | 104.1448 | 28.4233 | 118.4498 | 115.2838 | 124.5633 | 119.4323 | 4.717117 | 180.131 | 127.2926 | 188.4279 | 165.2838 | 33.16191 | 191.4629 | 175.9389 | 186.9105 | 184.7707 | 7.98014 | 259.4978 | 251.31 | 217.7948 | 242.8675 | 22.09622 | 103.3952 | 107.1179 | 157.3144 | 122.6092 | 30.1132 | 80.15284 | 61.57205 | 94.4214 | 78.71543 | 16.47178 |
| 0.1 | 19.68341 | 19.24672 | 20.17467 | 19.7016 | 0.464241 | 22.58734 | 23.79913 | 23.99563 | 23.4607 | 0.762709 | 24.50873 | 24.22489 | 27.13974 | 25.29112 | 1.607228 | 28.53712 | 49.06114 | 49.95633 | 42.5182 | 12.11624 | 49.31223 | 48.82096 | 49.67249 | 49.26856 | 0.42744 | 52.0524 | 50.20742 | 52.78384 | 51.68122 | 1.32771 | 58.16594 | 56.36463 | 59.12664 | 57.88574 | 1.402162 | 136.2445 | 118.3843 | 144.869 | 133.1659 | 13.50809 | 96.34279 | 93.19869 | 96.44105 | 95.32751 | 1.844267 | 85.53493 | 60.33843 | 93.19869 | 79.69068 | 17.19202 |
| 0.05 | 17.65284 | 17.22707 | 18.16594 | 17.68195 | 0.470109 | 20.5131 | 21.77948 | 20.84061 | 21.0444 | 0.657323 | 21.25546 | 22.19432 | 25.0655 | 22.83843 | 1.985009 | 25.3821 | 37.42358 | 37.67467 | 33.49345 | 7.02576 | 38.11135 | 37.89301 | 40.67686 | 38.89374 | 1.548077 | 40.42576 | 40.79694 | 40.86245 | 40.69505 | 0.235498 | 47.21616 | 48.02402 | 49.98908 | 48.40975 | 1.426139 | 129.1921 | 121.1681 | 111.1572 | 120.5058 | 9.03569 | 94.25764 | 93.3952 | 89.25764 | 92.30349 | 2.6728 | 88.31878 | 60.53493 | 93.3952 | 80.74964 | 17.68949 |
DTNB - keratinase activity
| 0 | 10 | 20 | 30 | 12h | 24h | 36h | |||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | Q7 | KU3 | DTT | Control | Q7 | KU3 | DTT | Control | Q7 | KU3 | DTT | Control | Q7 | KU3 | DTT | Control | Q7 | KU3 | DTT | Control | Q7 | KU3 | DTT | Control | Q7 | KU3 | DTT |
| 0.6146 | 1.555 | 0.4899 | 0.611 | 0.9908 | 0.5272 | 0.6065 | 0.9325 | 0.4539 | 0.6282 | 0.7674 | 0.3964 | 0.6234 | 0.4808 | 1.001 | 0.6234 | 0.4454 | 0.8526 | 0.6234 | 0.4909 | 0.7266 | |||||||
| 1.562 | 0.4374 | 1.022 | 0.5017 | 0.9601 | 0.4024 | 0.7681 | 0.4046 | 0.4885 | 0.9504 | 0.3937 | 0.772 | 0.5098 | 0.7243 | ||||||||||||||
| 1.554 | 0.4943 | 1.062 | 0.4645 | 0.8375 | 0.4508 | 0.7849 | 0.3934 | 0.4949 | 0.9983 | 0.4041 | 0.7457 | 0.4961 | 0.7398 | ||||||||||||||
22nd of September
500 µL of an appropriate dilution of enzyme (non-ultrafiltrated crude enzyme/dilutes) was added to 2500 µL of glycine-NaOH casein (0.05，0.1，0.5，1% w/v) in tris buffer (50 mmol/L, pH 7.5) and the mixture was incubated at 37°C. The solution after incubation was stored in -20℃.
24th of September
centrifuge KU3-casein samples & blank (rtp, 12000g, 2min)
Measure absorbance (A450) was using a spectrophotometer.
26th of September
1. KU3-36h samples & control: centrifuge (rtp, 12000g, 2min), terminated reaction by filtering through a 0.22um filter.
0.5ml filtrated solution add 2.5ml Na2CO3 & 0.5ml Folin reagent, rtp incubate 30min.
Measure absorbance (A450) with a spectrophotometer.
Microscopic morphology of hair
27th of September
1. Transfection of pCAMBIA-Cry7Ca1-eGFP vector through Carbon dots nanocomposite (CDP) to Nicotiana benthamiana
1. Coupling CDP with plant expressing vector
- Mix the following ingredients:
MES buffer(50X) 10ul
CDP(50X) 10ul
DNA(237ng/ul >10ng/ul final) 22ul
10% glycerol 25ul
ddH2O 433ul
total 500ul
- Gently mix and incubate at 37℃ for 30min.
2. Brush the mixture gently on the leaf of Nicotiana benthamiana(leaf length>10cm, Growing well), mark the area of brushing
3. Put the plant back in the light incubator(28℃，12h light 12h dark), repeat the process for four days at 3:00pm each day(1/4)
28th of September
1. Transfection of pCAMBIA-Cry7Ca1-eGFP vector through Carbon dots nanocomposite (CDP) to Nicotiana benthamiana(3/4)
29th of September
1. Transfection of pCAMBIA-Cry7Ca1-eGFP vector through Carbon dots nanocomposite (CDP) to Nicotiana benthamiana(4/4)
2. Fluorescence microscope to test the GFP, Negative result reason: CDP denatured
