Team:SZ SHD/Lab notes July
Lab Notes For July
7. 5
Selecting colonies of TOP 10 to inoculate and culture
- Participants: Amanda, Cindy, Emma, Jason, 蒋束修, Leonard, Mike
From the agar petri dishes yesterday, we used inoculation needles to collect TOP 10 cells and cultured them in liquid LB growth medium. Then we waited for the cultures to grow and develop. This would hopefully improve the efficiency and rate of success when we undergo shaking bacterial tomorrow.
Formula for liquid LB growth medium:
- 10g/L tryptone
- 5g/L yeast extract
- 10g/L sodium chloride
- 15g/L agar powder
- Variable volume of water
Procdures to prepare liquid LB growth medium:
Mix tryptone, yeast extract, and sodium chloride.
Then add the quantity of water (depending on the mass of solute).
Sterilize the solution at 121°c for 20 minutes.
General procedures:
- Add 5ml of liquid LB growth medium to ____
- Inoculate TOP10 cells using a pippette tip (or add 10-50 μl of bacteria solution if the cells are in a solution)
- Add 50 μg/mL of kanamycin (5μl)
- Incubate the culture at 37°c overnight while constantly shaking
7. 7
TOP 10 plasmid extraction and Measurement of its concentration and purity
- Participants: Amanda,Emma, Jason, 蒋束修, Leonard, Mike
Process
1. Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).
2. Harvest 2.5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15- 25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)
3. Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain. Note: No cell clumps should be visible after resuspension of TIANprep Mini Plasmid Kit Handbook | 4 the pellet, otherwise incomplete lysis will lower yield and purity.
4. Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 8 times. Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
5. Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.
6. Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
7. Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.
8. Repeat Step 7.
9. Centrifuge for an additional 5 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW. Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.
10. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 100 μl ddH2O (A little bit more, may use 80ul next time)to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
Results
| PET28a--T10 | Jason: KerBIER15 |
Amanda: KerBIER15 |
Emma: KerBteQ7 |
Mike： KerAvDZ50 |
蒋束修: KerBIMKU3 |
Leonard: KerBIMKU3 |
| Concentration: ug/ml |
1. 65.88 2. 59.980 |
1. 43.95 2. 33.43 |
1. 18.75 2. 17.63 |
1. 18.75 2. 17.63 |
1. 29.17 2. 17.05 |
1. 27.93 2. 22.94 |
| Purity: A260/A280 |
1. 1.820 2. 1.839 |
1. 1.820 2. 1.839 |
1. 1.86 2. 1.85 |
1. 1.86 2. 1.85 |
1. 1.901 2. 1.918 |
1. 1.950 2. 2.016 |
Sequencing
We sent the plasmid obtained that day to sequence. Results were received the next day.
Preservation of bacteria
Collect 500 µL of bacterial solution and 500 µL of 50 % concentration glycerol, place them in -80 °C fridge for long term preservation. (2 copies for each)
7.8
Transformation of plasmid to E.coli BL21 (DE3)
Procedure:
1. Take 50 µL of E.coli BL21 (DE3) cells, mix gently with 5 µL target plasmid (4 types of plasmid, place it on the ice bath for 25 min. *Make sure to defrost E.coli with ice bath and preheat waterbaath before hand.
2. Heat shock at 42 °C for 90 s in the water bath, place it immediately in the ice bath for another 2 min. DO NOT SHAKE.
3. Add 300 µL LB broth (not containing antibiotics), put it in the shaker to recover at 200 rpm, 37 °C for 60 min.
4. Centrifuge at 5,000 rpm for 1 min, keep 100 µL of supernatant and resuspend the bacteria. Coating on the LB broth.
5. Place upside down to incubate at 37 °C for 16 hrs in the incubator.
Material:
BL 21 (DE3), ice bath, water bath, 1.5 mL centrifuge tube, LB broth, shaker, centrifuge, incubator.
Results:
Three of four plasmids that were sended to sequencing failed to be sequenced. We suppose the reason is the low concentration. Thus we carry out PCR on these three plasmid and plan to test them again.
Sequencing Results (sent on July the 7th):
The table above shows the results of sequencing from the plasmid we collected on the 7th. Only sample PET28a-kerBIER-15 showed positive results, the rest remained negative. We suspect that the negative results were caused by low concentrations of plasmid inside our samples. Low concentrations could have lead to the plasmid being undetected. We have decided to redo the experiment and hopefully raise the concentration by polymerase chain reaction to increase the number of deoxyribonucleic acid
7.9
PCR
1. Mix up the recipe
- 25µl 2x T5 super PCR mix
- 2 µL forward primer
- 2 µL reverse primer
- To 50 µL (21 µL) ddH2O
- 50 µL in total
2. Prepare a new LB medium plate(solid) which contains antibiotics,,and label the plate and PCR tube
3. Use sterilized toothpick to obtain a single bacteria colony on the transformation plate. Draw a line on the LB plate on a copy of the original colony, and immense in the PCR tube contains the PCR recipe respectively to the label. Rotate the toothpick and throw it to the waste tank.
4. Repeat 1-3 for different bacteria colony
5.
Electropherosis
1. Gel recipe
- 50 ml TAE
- 0.5 g agar
- 5 µL Gel stain
2. Use a microwave oven to heat the solution for about 90 seconds. Note that turn the medium high heat to medium fire when solution start fizzing.
3. Pull out the solution quickly to the electrophoresis plate.
4. Wait for the solidification.
5. Drop 5 µL plasmid solution after PCR.
Result:
=Marker Z501234 Q7123344 U3 1234 Marker Q7 1234 U3 123 Z50 12
The results obtained from our electrophoresis test matched our initial hypothesized results prior to the experiment. All of the bands fell between the 2000bp and 1000bp range. This meant that all the DNA were the correct length. Hence, proving that our TOP 10 cells have the plasmid which enables them to produce keratinase.
Sequencing
We will send the plasmids for sequencing and verify the length of the bands for further experiments.
Selecting colonies of BL 21 to inoculate and culture
General procedures:
- Add 5ml of liquid LB growth medium to brown medium 2
- Inoculate BL21 cells using a pipette tip (or add 10-50 µl of bacteria solution if the cells are in a solution)
- Add 50 µg/mL of kanamycin (5μl)
- Incubate the culture at 37°D overnight while constantly shaking
Add 25 µL of 2x T5 Super PCR master mix, 2 µL of forward and reverse primer, add ddH2O up to 50 µL in total. Dip the pipette into the bacterial solution to extract enough culture and put it into the PCR tube, repeat twice for every type, 8 in total. Run the PCR program to replicate enough amount of plasmid for electropherosis later on.
Result:
Further steps done on electropherosis has shown the length between 1000 bp to 2000 bp, which indicates the approximately correct length compared to our target plasmid.
KerAvDZ50: 1217 bp
KerBIER15: 1175 bp
KerBIMKU3: 1202 bp
KerBteQ7: 1214 bp
Store the bacteria in the -80 °C fridge.
7.11
Prepare 800 mL liquid LB broth and 400 mL solid LB growth medium and sterilized under 121 °C for 20 min.
Reesults
Sequencing of the plasmids showed positive results and preliminary IPTG induced expression would be done afterwards. 5 mL of each bacteria culture was prepared and OD value measured between 0.4-0.6 in advance. Store at 4 °C overnight.
7.12
Due to the low OD valued determined yesterday, only 4 samples (2 for KerBteQ7-Emma, 1 KerAVDZ50-Mike and 1 KerBIER15-Jason) out of 7 showed OD value between 0.4 and 0.6, which is appropriate to use. Incubate the rest of bacteria culture for 7 min and determine the OD value to ensure it is in between the proper value.
IPTG induction
Concentrations for IPTG induced expression:
KerAVDZ50: 0.67139 mM (160 µL/mL)
Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50. (n.d.). International Journal of Biological Macromolecules, 92, 299–315.
KerBIER15: 0.4196 mM (0.01% w/v)
Tiwary, E., & Gupta, R. (2010). Extracellular Expression of Keratinase from Bacillus licheniformis ER-15 in Escherichia coli. Journal of Agricultural and Food Chemistry, 58(14), 8380–8385.
KerBIMKU3: 0.4 mM
Radha, S., & Gunasekaran, P. (2007). Cloning and expression of keratinase gene in Bacillus megateriumand optimization of fermentation conditions for the production of keratinase by recombinant strain. Journal of Applied Microbiology, 103(4), 1301–1310.
KerBteQ7: 5 mM
Zaraî Jaouadi, N., Rekik, H., Ben Elhoul, M., Zohra Rahem, F., Gorgi Hila, C., Slimene Ben Aicha, H., Badis, A., Toumi, A., Bejar, S., & Jaouadi, B. (2015). A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process. International Journal of Biological Macromolecules, 79, 952–964.
SDS-PAGE
1. Dilute Pre-Cassettle Gel Running Buffer 20x
2. Mix the loading and bacteria solution (total volume is about 40uL) and heat to 95 degrees for 10min
3. Fix protein gel to the machine
4. Add pure water to the middle of the machine to check the sealing( if there's no water on the bottom of the machine after 10min or there's no water colume after lifting it up, the machine is OK to use)
5. Heat the marker to 95 degrees for 1min
6. Pour out the pure water and add SDS-PAGE buffer to the middle until the solution overflows
7. Take out the model of pits gently
8. Drop the bacterial solution and marker
9. Cover the lid and connect the circuit
10. Start the machine at 80v for 30min, then change to 120v for 1h (Note that add buffer every 10min to prevent the gel from drying out)
7.13
OD Value
IPTG induction
KerAVDZ50
Mike
【11:12】
1.Add 13ul IPTG(1mol/L) to 21mL bacteria solution to make the solution with IPTG 0.67139mmol/L in the solution
2.Measure the mass of the centrifuge tube
3.Put the centifuge tube to the 37 °C shaking incubator for 4 hours
【15:12】
(SDS-PAGE run protein
1. Dilute Pre-Cassettle Gel Running Buffer 20x to 1x (30ml buffer in 600ml solution)
2. Mix the loading and bacteria solution (total volume is about 40uL) and heat to 95 degrees for 10min
3. Fix protein gel to the machine
4. Add pure water to the middle of the machine to check the sealing( if there's no water on the bottom of the machine after 10min or there's no water colume after lifting it up, the machine is OK to use)
5. Heat the mark to 95 degrees for 1min
6. Pour out the pure water and add SDS-PAGE buffer to the middle until the solution overflows
7. Take out the model of pits gently
8. Drop the bacterial solution and marker
9. Cover the lid and connect the circuit
10. Start the machine at 80v for 30min, then change to 120v for 1h (Note that add buffer every 10min to prevent the gel from drying out) add buffer every ten minutes)
4.Centrifuge at 4 °C（10000g 10min） collect bacteria
(mass of bacteria:0.124g)
5.Add Tris Buffer to regulate the PH value to 7.5（5ml/0.5g，1.24ml)
6.Ultrasonication（put mixture of ice and water to beaker and put the centrifuge tube in it）(undetermined)
7.Centrifuge and take the supernate（15000g 20min）(undetermined)
【9:30】
1.Add 22.73ul IPTG(1mol/L) to 32.5mL bacteria solution to make the solution with IPTG 0.67139mmol/L in the centrifuge tube
2.Measure the mass of the centrifuge tube
3.Put the centifuge tube to the 37 °C shaking incubator for 12 hours
KerBIMKU3
Leonard 蒋束修
【11:05】
1.Add 7.4ul IPTG(1mol/L) to 18.5mL bacteria solution to make the solution with IPTG 0.4mmol/L in the centrifuge tube
2.Measure the mass of the centrifuge tube
3.Put the centifuge tube to the 37 °C shaking incubator for 4 hours
【15:05】
(SDS-PAGE run protein
- Dilute Pre-Cassettle Gel Running Buffer 20x
- Mix the loading and bacteria solution (total volume is about 40uL) and heat to 95 degrees for 10min
- Fix protein gel to the machine
- Add pure water to the middle of the machine to check the sealing( if there's no water on the bottom of the machine after 10min or there's no water colume after lifting it up, the machine is OK to use)
- Heat the mark to 95 degrees for 1min
- Pour out the pure water and add SDS-PAGE buffer to the middle until the solution overflows
- Take out the model of pits gently
- Drop the bacterial solution and marker
- Cover the lid and connect the circuit
- Start the machine at 80v for 30min, then change to 120v for 1h (Note that add buffer every 10min to prevent the gel from drying out) add buffer every ten minutes)
4.Centrifuge at 4 °C（10000g 10min） collect bacteria
(mass of bacteria: 0.211g)
5.Add Tris Buffer to regulate the PH value to 7.5（5ml/0.5g，2.11ml）
6.Ultrasonication（put mixture of ice and water to beaker and put the centrifuge tube in it）(undetermined)
7.Centrifuge and take the supernate（15000g 20min）(undetermined)
KerBteQ7
Emma
1.Add 250ul IPTG(1mol/L) to 50mL bacteria solution in the centrifuge tube to make the solution with IPTG 5mmol/L in the centrifuge tube
2.Measure the mass of the centrifuge tube
3.Put the centifuge tube to the 37 °C shaking incubator for 12 hours
KerBIER15
Jason Amanda
1.Add 6 IPTG(1mol/L) to 15mL bacteria solution to make the solution with IPTG 0.4mmol/L in the centrifuge tube
2.Measure the mass of the centrifuge tube
3.Put the centifuge tube to the 37 °C shaking incubator for 16 hours
7.14
Wash the induced bacteria
1. Centrifuge the induced bacteria solution at 8000rpm for 5min at 4 degrees 2. Suck supernatant out of the tube 3. Add 5mL PBS and resuspend 4. Repeat 1-3 steps 2-3 times 5. Weigh the mass of remaining ppt, then add PBS(10ml/g)
Sonication
1. Place the tube that contains washed bacteria solution in a beaker with ice.
2. Place the beaker on the platform of the sonocation machine
3. Adjust the position of platform to make sure the probe contacts the bacteria solution but doesn't contact the bottom of tube.
4. Take a picture of tube to compare the clarity of the bacteria solution before and after sonication.
5. Set the program with opening 6s and closing 6s, 3M in total
6. Test the position and noise before start the program
7. Spary ethanol to the probe if the temperature is too high, then wipe out the remaining ethanol by tissue.
Centrifugation
1. Suck out the most bubbles
2. Add Triton(10x) to 1x and pipette up and down
3. Place in the ice for 20-30min
4. Centrifuge at 15000g for 30min
SDS-PAGE
1. Dilute Pre-Cassettle Gel Running Buffer 20x
2. Mix the loading and sample (total volume is about 40uL) and heat to 95 degrees for 10min
(Note that the sample we took this time is the supernatant, ppt(resuspend by ddH2O) and bacteria solution of 4 bacteria strains before induction respectively.)
3. Fix protein gel to the machine
4. Add pure water to the middle of the machine to check the sealing( if there's no water on the bottom of the machine after 10min or there's no water colume after lifting it up, the machine is OK to use)
5. Heat the marker to 95 degrees for 1min
6. Pour out the pure water and add SDS-PAGE buffer to the middle until the solution overflows
7. Take out the model of pits gently
8. Drop the bacterial solution and marker
9. Cover the lid and connect the circuit
10. Start the machine at 80v，120mA，for 30min, then change to 120v for 1h (Note that add buffer every 10min to prevent the gel from drying out)
11. Shake the gel with Fast Protein Stain
Result
7.16
OD value
7.17
7.18
【0:40】SDS-PAGE. Shake the gel with Fast Protein Stain at 60rpm for 25min.
【1:15】Photo was taken：
Prestained protein marker, Z50 supernatant, R15 supernatant, KU3 supernatant, Q7 supernatant，x, x, x, R15 precipitate, x, R15 supernatant, Q7 precipitate, Q7 supernatant, marker
【20:00】Preparation of LB liquid media.
【22:00】 Inoculation of 4.5 mL BL21 into 150mL LB broth (in 4 x 250mL conical flask) for storage. The time of cultivation should be 15h (~ 1pm, 19th Jul)
Inoculation of 3mL (1.5%) BL21 (kerBIER15) into 200mL LB broth for induction. The time of cultivation should be 18h (~ 4pm, 19th Jul).
7.19
【3: 00】Inoculation of 3mL (1.5%) BL21 (kerBteQ7; kerAvDZ50) into 200mL LB broth for induction. The time of cultivation should be 12h (~ 3pm, 19th Jul).
The incubator was not shaking, we checked the OD value (~0.1).
【13:00】 Optical Density Test
IPTG induced expression done to KerBIER15
1. 【15.05】
OD value of 200 mL bacterial solution KerBIER15 measured. 0.7979 after blank subtraction.
2. 【15.35】
80 µL of 1 M IPTG solution added to 200 mL of KerBIER15 bacterial solution to balance the final concentration to 0.4 mM. Incubate at 37 °C for 16 hrs as reference states. [condition of shaker: 37 °C, 220rpm] Collect the bacterial culture at 07.35 20th July.
IPTG induced expression done to KerBteQ7 and KerAvDZ50
1. 【17.36】
OD values of KerAvDZ50 and KerBteQ7 are 0.7760 and 0.8552 respectively.
2. 【18.00】 IPTG added
134 µL of IPTG added to KerAvDZ50 to balance the final concentration to 0.67139 mM; 1000 µL of IPTG added to KerBteQ7 to balance the final concentration to 5 mM. Incubate at 16 °C for 14 hrs. [Condition: 16 °C, 220 rpm] Collect the bacterial culture at 8.00 20th July.
7.20
Preparing Four Solutions For Purification
ELB: PBS 5Oml, imidazole(MW:68.08g/mol) 0.03404g
WB: PBS 5Oml, imidazole 0.0851g
EB: PBS 5Oml, imidazole 0.851g
EB2: PBS 50ml, imidazole 1.1914g
PH = 7.4- 8.0
Sonication
1. Place the tube that contains washed bacteria solution in a beaker with ice.
2. Place the beaker on the platform of the sonocation machine
3. Adjust the position of platform to make sure the probe contacts the bacteria solution but doesn't contact the bottom of tube.
4. Take a picture of tube to compare the clarity of the bacteria solution before and after sonication.
5. Set the program with opening 6s and closing 6s, 3M in total
6. Test the position and noise before start the program
7. Spary ethanol to the probe if the temperature is too high, then wipe out the remaining ethanol by tissue.
40% intensity for 15 minutes (at six seconds interval)
[6 sec sonication + 6 sec pause] x n times
Bacterial after induction
6250ul Resin
After centrifugation about 5ml resin + 25ml ELB
7.21
Washing Ni-Resin
All materials were kept in icebox to ensure a low temperature (4°C)
Gently shake the Ni-Resin container before using to prevent layering
Add Ni-Resin into a separate tube (to make sure a about 6ml was added)
Centrifugate at 1000rpm *4°C for 2min, then discard the superantant (low-speed centrifugation)
-> Add ELB (5 times of Ni-Resin volume) per tube, pipette up and down, then centrifugate at 3000rpm/700g for 2min and discard ELB
Above procedure to be done twice
Table 1: Procedure
Take a sample of 20ul superantant from a total of 25ml for SDS-PAGE later
Add same volume of ELB(25ml) to the superantant(25ml), and mix them together(30min)
Add the pre-washed Ni-Resin into the above liquid, suspend the Resin, pipette up and down every half hour(when you see Resin at the bottom) for about 2-3hrs at 4°C
Table 2
Centrifugate at 700g for 2min, and transfer 20ul superantant to another 1.5ml tube [on ice] for SDS-PAGE later.
->Add EB(same volume as Resin) to wash off protein(add 5ml EB to 5ml Resin), pipette up and down to suspend Resin, then set it aside for 3-5min to let the Resin sink down, centrifugate at 700g for 2min at 4°C, and transfer (pipette) the superantant to a new tube
Repeat the above step for three times (in seperate tubes), and take a sample of 20ul superantant each round for SDS-PAGE later.
SDS-PAGE result
Figure. SDS-PAGE stained with Fast Protein Stain lane1. Protein molecular marker; lane2. Empty PET-28a vector; lane3. Crude enzyme (kerAvDZ50); lane4. Supernatant obtained after 3h Ni-NTA incubation; lane5. Supernatant obtained after 1st protein wash; lane6. Supernatant obtained after 2st protein wash; lane7. Empty PET-28a vector; lane8. Crude enzyme (kerBteQ7); lane9. Supernatant obtained after 3h Ni-NTA incubation; lane10. Supernatant obtained after 1st protein wash; lane11. Empty PET-28a vector; lane12. Crude enzyme (kerBlER15); lane13. Supernatant obtained after 3h Ni-NTA incubation; lane14. Supernatant obtained after 1st protein wash; lane15. Supernatant obtained after 2st protein wash
This is not an expected result that we hope to get. Lane 2 and 3 inclusion body
7.22
Prepare solutions
150ml stripping buffer (50mM NaH2PO4, 300mM NaCl, 100mM EDTA, PH 8.0)
225ml deionized water
75ml NiSO4 (1.971375g NiSO4*6H2O,75ml PBS)
150ml PBS
Recycle Ni-Resin
1. Wash with stripping buffer
2. Wash the Ni-Resin with deionized water
3. Wash the Ni-Resin with NiSO4 dissolved in water
4. Wash the Ni-Resin with PBS (to balance the Ni-Resin)
5. Store the balanced Ni-Resin with 20% ethanol
Inoculate 3% bacterium culture in 200ml of liquid medium [500ml flask] at 37°C 220rpm for 4hrs
Check for OD value
| Name of bacteria | KerBteQ7 | KerAVDZ50 |
| Od value | 0.6723 | 0.5029 |
Induction
Add 1000ul 1M IPTG to Q7 and 134.278ul 1M IPTG to Z50
Incubate at 220 rpm 22°C overnight for 12hrs
7.23
Sonification and purification, steps are the same as July 20th and 21th
Supernatant samples for SDS-PAGE are stored in -20°C
7.24
SDS-PAGE and recycle Ni-Resin, steps are the same as July 21th and 22thSDS-PAGE result
KU3 0.162g
R15 0.246g
7.26
1. Made solutions needed(god said let there be WB,EB,ELB and LB, and there were WB, EB, ELB, and LB)
2. Sneaked to other groups' camps and "asked gently for" things we need
3. Crudely destroyed habitats of bacteria in our solution and 50 ml centrifugual tubes
4. Give new birth to dying bacteria(Z50 and Q7)
7.27
Purification of protein
SDS-page results
Figure. SDS-PAGE stained with Fast Protein Stain lane0. Protein molecular marker; lane1. kerAvDZ50 Bacteria before induction; lane2. Crude enzyme (kerAvDZ50); lane3. Supernatant obtained after filtration through Ni-Resin ;lane4. Supernatant obtained after Wash Buffer; lane5. Supernatant obtained after Elute Buffer; lane6. KerBTEQ7 bacteria cell before induction; lane7. Crude enzyme (kerBTEQ7); lane 8. Supernatant obtained after filtration through Ni-Resin; lane9. Supernatant obtained after WB; lane10. Supernatant obtained after EB; lane11. Empty thing
Figure. SDS-PAGE stained with Fast Protein Stain lane1. Protein molecular marker; lane2. kerBIER15 Bacteria before induction; lane3. Crude enzyme; lane4. Supernatant obtained after filtration through Ni-Resin ;lane5. Supernatant obtained after Wash Buffer; lane6. Supernatant obtained after 1st Elute Buffer; lane7. Supernatant obtained after 2st Elute Buffer; lane8. Supernatant obtained after 3rd Elute Buffer
Bacteria expansion(1. KerBIER15, KerBIMKU3)
7.28
1. purification of protein(KerBIER15, KerBIMKU3)
2. ultrafiltration of protein(KerAVDZ50, KerBTEQ7)
3. bacteria expansion(KerBTEQ7)
SDS Results
Figure. SDS-PAGE stained with Fast Protein Stain lane1. Protein molecular marker; lane2. KerBIMKU3 Bacteria before induction; lane3.KerBIER15 Bacteria before induction; lane4. Supernatant of dilute KerBIMKU3 Bacteria obtained after filtration through Ni-Resin; lane5. Supernatant of dilute KerBIMKU3 Bacteria obtained after Wash Buffer; lane6. Supernatant of dilute KerBIMKU3 Bacteria obtained after Elute Buffer; lane7. Supernatant of dilute KerBIER15 Bacteria obtained after filtration through Ni-Resin ; lane8. Supernatant of dilute KerBIER15 Bacteria obtained after Wash Buffer; lane9. Supernatent of dilute KerBIER15 Bacteria obtained after Elute Buffer; lane10. Supernatent of KerBIMKU3 Bacteria obtained after filtration through Ni-Resin; lane11. Supernatent of KerBIMKU3 Bacteria obtained after Wash Buffer; lane12. Supernatent of KerBIMKU3 Bacteria obtained after Elute Buffer; lane13. Supernatent of KerBIER15 Bacteria obtained after Filtration through Ni-Resin; lane14. Supernatent of KerBIER15 Bacteria obtained after Wash Buffer; lane15. Supernatent of KerBIER15 Bacteria obtained after Elute Buffer.
7.29
1.
Collecting bacteria Q7 600mL
Collecting bacteria through centrifuge
8000rpm, 4℃, 5min
50mL centrifuge tube, dispose supertanant
Resuspend bacteria 5 mL pbs (repeat twice)
Weigh bacteria 3.8g
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)
2.
Electrophoresis
3ul of marker
10ul of bacteria solution before induction (KU3), bacteria solution before induction (R15), KU3 (dilute) supertanant, WB (KU3) (dilute), EB (KU3) (dilute), R15 supertanant (dilute), R15 WB (dilute), R15 EB (dilute), KU3 supertanat, KU3 WB, KU3 EB, R15 supertanat, R15 WB, R15 EB
3.
Culture 600 mL of R15 bacteria for 4h at 37℃ and 220 rpm
Check OD and induce with IPTG
7.30
1. Electrophoresis
2. Collecting bacteria through centrifuge 8000rpm, 4℃, 5min
3. Purification (Q7)
4. Condense (Q7)
5. Culture 600 mL of Q7 bacteria for 4h at 37℃ and 220 rpm
Check OD and induce with IPTG
7.31
1. Collecting bacteria through centrifuge
8000rpm, 4℃, 5min
50mL centrifuge tube, dispose supertanant
Resuspend bacteria 5 mL pbs (repeat twice)
Weigh bacteria 3.0
2. Purification
(check precious procedures on purification)
3. Electrophoresis
Preparation for electrophoresis: take 30 μL of bacterial solution before induction, supernatant after sonification, solution after purification with wash buffer, solution after elusion buffer, purified Q7 protein, and purified R15 protein.
Add 30mL of loading into each sample
Heat the sample at 95℃ for 10min
Trial 1: 90V 100mA 50min
Trial 2: 100V 100mA 45min
Conclusion: time is not enough for recording, it is difficult to observe information and make conclusion.
4. Culture 600 mL of R15 bacteria for 4h at 37℃ and 220 rpm
Check OD and induce with IPTG