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Our experiment process is separated into two stages—Beijing and Shenzhen. In Beijing, due to restriction in facilities, we didn’t get much solid progress or systematic results, however, the many days we spent in the Beijing lab laid good foundation for us in terms of our skills in the lab, and it also allowed us to familiarize with our proteins, bacteria and other conditions through trial and error, which allowed us to complete the most essential parts of our experiments and get the important results in a relatively short amount of time.

Beijing (July 22th-Oct 26th)

We were planning to use the lab in Tsinghua University, however, due to the covid-19 situation, Tsinghua University was not opened to the public. Our instructor connected a Lab in Beijing so that we could start the experiment.

July 22th

  1. Prepare 500ml antibiotic-free LB liquid and store it in a refrigerator at 4°C
  2. Cultivate LCC in a 60ml LB liquid
  3. Extract the LCC plasmid in the small Erlenmeyer flask and store it in the refrigerator at 4°C

July 23th

transform pET28a-LCC plasmid into BL21 competent cell

July 24th

The fragile BL21 competent cell with pET28a-LCC died, we realized that it died because it was stored in -20°C fridge instead of -80°C because the -80°C fridge was inaccessible at the time.

July 27th Monday

  1. Transform competent cells: Transform 100µl competent cells (into 600ul bacterial solution), and then screen for selection.
  2. At one o'clock in the afternoon, a tube of competent cells in ice (total) was retrieved from the middle school attached to Tsinghua University, 100 µl was taken out of it, and transformation began.
  3. Add 1µl plasmid (pET28a-LCC) to the tube containing competent cells.
  4. place the tube containing competent cells with 1µl plasmid of pET28a-LCC in the ice box for 30min.
  5. place the tube containing competent cell into the hot water bath for 1 minute for a heat shock.
  6. Place the tube containing competent cell with 1µl pET28a-LCC plasmid back into the ice box.
  7. Complete the following operation on a clean bench; add 500ul antibiotic-free petri dish to the the tube containing competent cell with 1µl pET28a-LCC plasmid.
  8. Insert the the tubes containing competent cell with 1µl pET28a-LCC plasmid in the float and put it in a shaker to recover for one hour.
  9. Take out the tube containing transformed competent cells and apply it to four kan-petri dishes for screening. (There are four in total, one of it has 200µl and three has 100ul)
  10. Put the four kan-petri dishes in an incubator.

Expansion: LCC, BliS, LALBA, LTF have been amplified respectively

July 28th Tuesday

The original plan was to expand the untransformed competent cells (remaining yesterday), but since the cells are still in the logarithmic phase, there is no need for further expansion. We then decided to use the bacterial liquid we had to make competent cells

Transform competent cells: Transform 100µl competent cells (into 600ul bacterial solution), and then screen for selection.

  1. Add 1µl plasmid (pET28a-LCC) to the tube containing competent cells
  2. place the tube containing competent cells with 1µl plasmid of pET28a-LCC in the ice box for 30min.
  3. place the tube containing competent cell into the hot water bath for 1 minute for a heat shock.
  4. Place the tube containing competent cell with 1µl pET28a-LCC plasmid back into the ice box.
  5. Sterilize three conical flasks and three centrifuge tubes for the experiment. Then sterilize 13 centrifuge tubes for future use.
  6. Take out a bottle of Kan-LB medium, and add 20ml into each of the three centrifuge tubes.
  7. Prepare two 2ml centrifuge tube, add 2ml antibiotic-free medium in to one and add 2ml with competent cells in to the other one.

Perform OD analysis.

  1. First put the two cuvettes into the luminometer for peeling
  2. Then add the Kan-LB medium and competent cells to the two dishes for photometry. The result is OD=0.663. We gain the conclusion that the competent cells with pET28a-LCC plasmid are still roughly in the logarithmic period (OD value 0.4-0.6).

Self-made competent cells
Since the cell is in good condition, the expansion experiment is suspended, we then starts to make the competent cells: Untransformed competent cells (which have returned to normal form) are made into competent cells by calcium chloride.

  1. Perform OD analysis. First put the two cuvettes into the luminometer for peeling, and then add the anti-LB and competent cells to the two dishes for photometry. We gained the conclusion that they are still in the logarithm period.
  2. Use a pipette to add 4 ml of glycerol to a 5 ml centrifuge tube on the ultraclean table.
  3. Measure 1.1 g of anhydrous calcium chloride, put it into a washed jar, and add 100 ml of deionized water.
  4. Use a pipette to add 4 ml of glycerol to a 5 ml centrifuge tube on the clean bench.
  5. Measure 1.1 g of anhydrous calcium chloride, add it into a washed jar, and then add 100 ml of deionized water.
  6. Sterilize the jar containing the medicine (sterilize together with the 13 centrifuge tubes in the expansion experiment).
  7. Divide the non-competent bacteria liquid into 11 2ml centrifuge tubes and store them in an ice box for freezing. (Its purpose is to keep the competent cells stable)
  8. Balance the 2ml centrifuge tubes by placing them into three groups of triangles and one group of symmetrical. Then, centrifuge the eleven tubes of bacteria liquid
  9. Dispense three centrifuge tubes of calcium chloride (12ml in total, 4ml per tube, we adjusted them to 4.5ml per tube in order to facilitate the operation of the pipette). Put them in an ice box to cool down.
  10. Eleven tubes of glycerin are separated at the clean bench
  11. At the clean bench, discard the waste liquid.
  12. Add 1 ml of calcium chloride in to the tubes containing bacteria and resuspend.
  13. Centrifuge the resuspended solution again, discard the liquid part, and retain the precipitate.
  14. Discard the waste liquid at the clean bench.
  15. Add 70ml calcium chloride to the bacteria and resuspend.
  16. Add the resuspended solution to the glycerin tube and store it in the refrigerator.

Extract plasmid:

  1. Take two tubes of Kanamycin from the -20 dgrees of Celsius fridge.
  2. Using the pipette tip, scratch the bacteria clumps (LCC and bLIS respectively) that were transformed yesterday in the petri dish gently. After the operation is completed, the pipette tips are respectively injected into two 50 ml centrifuge tubes of Kanamycin-LB liquid.
  3. Place the Kan-LB liquid containing the pipette tip and the bacterial clumps into a shaker for pee-culture.
  4. Divide the two 50 ml centrifuge tubes of Kanamycin-LB liquid with a monoclonal colony into three small tubes (15ml each)

July 29th Wednesday

Protein induced expression:

  1. Take out the LB broth from the autoclave.
  2. Put the three Erlenmeyer flasks in the washbasin to cool down for 5 minutes
  3. From the -20 dgrees of Celsius fridge, take out the Kanamycin and add 0.5 ml of Kanamycin to each Erlenmeyer flask.
  4. Add 5ml of pre-cultured bacterial solution to each of the Erlenmeyer flask and incubate for 4 hours at 37°C
  5. Place the two Erlenmeyer flasks in the shaker, set the temperature to 26 degrees Celsius, and wait for 10 minutes
  6. Add 250 microliters of IPTG in each of the two Erlenmeyer flasks.

Configure liquid medium:

  1. Prepare three conical flasks
  2. Add 500ml of deionized water into each conical flask
  3. Add 2.5 grams of yeast extract, 5 grams of sodium chloride and 5 grams of meringue into each conical flask.
  4. Sterilize these three Erlenmeyer flasks for one hour (Kraft paper, tin foil and rubber band cover the top of the Erlenmeyer flask)
  5. After sterilization and cooling, add 0.5ml of Kanamycin to each conical flask.

Transformation competent cell

  1. chipping ice from the -20 degrees Celsius fridge since there isn’t an ice maker in this lab.
  2. Take out the 3 tubes of the self-made competent cell from yesterday and take out the one we bought from the company out of the refrigerator from -80 degrees Celsius, and store them in the ice box temperately.
  3. Take out a tube of dry powdered bLIS plasmid (4 micrograms) from the refrigerator and put it in a centrifuge, then add 50 microliters of deionized water.
  4. On the clean bench (noted: do not touch the competent cells with hands), leave the competent cells in the ice box and let wait for 4 min until it is partly melted. Use a pipette to add 1-2 microliters of bLIS plasmid into the bacterial solution. (Repeat 4 times)
  5. Incubate the competent cell on ice for 30 minutes
  6. Heat the water bath at 42 degrees for 1 minute
  7. Place it in the ice box for 1 minute
  8. Add 500 μl of antibiotic-free LB liquid
  9. Add 200 microliters of competent cell to the LB medium
  10. Put the competent solution into the shaker to recover.

Save bacteria in case of failure of the experiment

  1. Seal the BL21 with pET28a-LCC plasmid with parafilm and put it in the refrigerator at -4 degrees Celsius.

Screening:

  1. Add 200 microliters of BL21 (with bLIS plasmid) on a petri dish containing solid Kan-LB medium using the Pipette .
  2. Put the screened solid LB medium into the shaker.

July 30th Thursday

separation of LCC protein:

  1. Prepare centrifuge tubes, scales, tube racks, and bacteria liquid with IPTG added yesterday Divide the LCC bacteria liquid into 50ml centrifuge tubes, each centrifuge tube has about 30ml bacteria liquid, use the scale to "peel" to ensure the same weight (there are 15 tubes in total at the end)
  2. Centrifuge the 15 tubes of bacteria liquid, (set-4000 rpm for 30 minutes)
  3. After 30 min, pour out the waste liquid and leave bacteria clumps at the bottom of the centrifuge tube.
  4. Dilute buffer: Tris-hydrochloric acid buffer-1.5Molar diluted 30 times to 50 micromoles per liter
  5. Add 30ml Tris-Hcl buffer ph8 in to each centrifuge tube, stir the solution using the tip of the pipette, then resuspend the bacteria clumps.
  6. Add Tris-HCL buffer with bacteria into a beaker, place the beaker in to the ultrasonic disruptor. Time set 15 minutes, set the machine to stop for six seconds after working for every six seconds.
  7. After the Ultrasonic disruption, add the solution back into the centrifuge tubes and centrifuge at 4000 rpm for 30 minutes ((in order to seperate the bacteria remains with the protein)
  8. After the centrifuge is done working, the precipitation are the bacteria remains and the supernatant is the protein wen need. Collect the supernatant into a beaker using a pipette
  9. Put a small piece of PET plastic sheet (we were expecting for degradation observable with naked eyes)
  10. Find a same beaker, add the same amount of water as much as the supernatant is. Then add a piece of PET plastic sheet at the same size as the one being put in the supernatant.
  11. Place the two beakers in a shaker at room temperature overnight.

July 31th Friday

We decided to do a DNA gel electrophoresis for the bLIS and LCC plasmids extracted on July 28th.

Materials needed:

  • Agarose 2g
  • TAE 100ml
  • DNA dye 10000x
  • DL5000 DNA marker 5µl
  • Purchased bLIS plasmid as a control

DNA gel electrophoresis

  1. Add 48ml TAE and 2g agarose in to a 100ml conical flask, and heating using microwave oven (15 seconds, maximum power)
  2. After it cools down, take the Gel Red dye out of the 4 degrees Celsius fridge, add 5µl into the gel
  3. Take the DNA loading dye and marker out of the -20 degrees Celsius fridge, and put it in an ice box for 15mins for it to melt slowly.
  4. Set up the equipment for DNA gel electrophoresis
  5. Prepare a PCR-8-Tube Strip, add the marker and samples in the order below:

    From left to right:

    • DNA Marker(5µl)
    • Purchased bLIS(10µl)
    • extracted bLIS(10µl)
    • extracted bLIS(10µl)
    • extracted LCC(10µl)
    • extracted LTF(negative control,10µl)
  6. Add DNA Loading dye 2µl into each tube of sample.
  7. Power up and wait for 4 hours
  8. Place the gel into the gel imager to see the result

August 3rd Monday

  1. Clean the required instruments
  2. Dilute the precast gel with tris-HCl buffer
  3. Add extracted 50µl LCC supernatent to 4 PCR tubes.
  4. Add 20 µl Tris-HCL buffer to each PCR tube.
  5. Add the loading buffer of the protein to the PCR tube, the ratio of the loading buffer to the protein solution is 1:5
  6. Centrifuge the liquid on the tube wall to the bottom of the tube in a centrifuge
  7. Put the protein in a water bath and set the temperature to 70 degrees Celsius
  8. Remove the seal under the configured protein gel, fix it on a shelf and close the lid to seal it.
  9. Dilute the SDS buffer with water, to get 1x SDS-PAGE buffer
  10. Pull out the comb and add buffer. (Both positive and negative electrodes are added), the negative electrode is added between the high and low glass,
  11. After setting up the apparatus, add the Protein marker and the prepared sample into the gaps on top of the gel
  12. Turn on the electrophoresis apparatus
  13. Set voltage 100V
  14. When all the protein samples reaches the area between the concentrated gel and the separating gel, switch the voltage to 120V
  15. After the samples reach the bottom of the instrument, turn off the electrophoresis instrument
  16. Take out the gel together with the glass and soak it in water, and pry open the glass with a tool/comb, leaving only the gel
  17. Cut off the strips without samples/markers
  18. Cut off a small corner on the right side of the gel, to distinguish the front and back
  19. Soak the gel in Coomassie Brilliant Blue dye, put it in a horizontal shaker, and shake for one hour
  20. Stop the horizontal shaker and recycle Coomassie Brilliant Blue back into the centrifuge tube
  21. Rinse the protein gel and petri dishes slowly at the tap,
  22. As there are still remaining Coomassie Brilliant Blue left on the gel in the petri dish, add the eluent (the eluent must be over the protein gel) and shake it from 3:40 to 4:40 in a horizontal shaker
  23. Slowly pour the eluent into the pool at 4:40 and rinse the protein gel slowly
  24. Add the new eluent into the petri dish (the eluent must be over the surface of the protein gel)

August 4th Tuesday

Since the SDS-PAGE didn’t went very well yesterday, we then decided to redo the experiment.

  1. Pre-culture BL21 with pET28a-LCC in the morning and clean the required instruments
  2. Dilute the precast gel with tris-HCl buffer
  3. Add extracted LCC supernatent to 4 PCR tubes. In order to control the protein density for each sample and make the density increase in a proportion,
    • add 70µl LCC supernatent in the first one
    • add 50µl LCC supernatent in the second one
    • add 25µl LCC supernatent in the third one
    • add 15µl LCC supernatent in the fourth one
  4. Add Tris-HCL buffer to each PCR tube until it reaches 70 µl.
  5. Add the loading buffer of the protein to the PCR tube, the ratio of the loading buffer to the protein solution is 1:5
  6. Centrifuge the liquid on the tube wall to the bottom of the tube in a centrifuge
  7. Put the protein in a water bath and set the temperature to 70 degrees Celsius
  8. Remove the seal under the configured protein gel, fix it on a shelf and close the lid to seal it.
  9. Dilute the SDS buffer with water, to get 1x SDS-PAGE buffer
  10. Pull out the comb and add buffer. (Both positive and negative electrodes are added), the negative electrode is added between the high and low glass,
  11. After setting up the apparatus, add the Protein marker and the prepared sample into the gaps on top of the gel
  12. Turn on the electrophoresis apparatus
  13. Set voltage 100V
  14. When all the protein samples reaches the area between the concentrated gel and the separating gel, switch the voltage to 120V
  15. After the samples reach the bottom of the instrument, turn off the electrophoresis instrument
  16. Take out the gel together with the glass and soak it in water, and pry open the glass with a tool/comb, leaving only the gel
  17. Cut off the strips without samples/markers
  18. Cut off a small corner on the right side of the gel, to distinguish the front and back
  19. Soak the gel in Coomassie Brilliant Blue dye, put it in a horizontal shaker, and shake for one hour
  20. Stop the horizontal shaker and recycle Coomassie Brilliant Blue back into the centrifuge tube
  21. Rinse the protein gel and petri dishes slowly at the tap,
  22. As there are still remaining Coomassie Brilliant Blue left on the gel in the petri dish, add the eluent (the eluent must be over the protein gel) and shake it until 5:20 in a horizontal shaker
  23. Slowly pour the eluent into the pool at 5:20 and rinse the protein gel slowly
  24. Add the new eluent into the petri dish (the eluent must be over the surface of the protein gel)

As the SDS is going on, we also started the next round of experiment Protein induced expression:

  1. Take out two bottles of liquid medium with Kanamycin from the refrigerator
  2. According to the ratio of 1:100, add the Add 5ml of BL21 with pET28a-LCC, add solution to each Erlenmeyer flask and incubate for 4 hours at 37°C
  3. Take out the Erlenmeyer flask from the shaker
  4. Prepare three cuvette
  5. At the clean bench, fill the two of the cuvettes with the bacteria liquid of BL21 with pET28a-LCC from two different conical flasks. And use antibiotic-free LB liquid to compare with.
  6. Defrost IPTG in the ice box
  7. Test OD value, the results were 0.462-control group, 0.611-will be induced expression group
  8. Add IPTG at a ratio of 1:1000 to the Erlenmeyer flask that will induce the expression group
  9. Put two Erlenmeyer flasks in the shaker, set the temperature to 26 degrees, and wait over night.

Prepare antibiotic-free LB liquid:

  1. Prepare two conical flasks and a measuring cylinder
  2. Add 2.5 grams of yeast extract, 5 grams of sodium chloride and 5 grams of peptone into each Erlenmeyer flask.
  3. Add 500ml of water into each Erlenmeyer flask and shake well
  4. Sterilize these two Erlenmeyer flasks for one hour (Using Kraft paper, tin foil and rubber band cover the mouth of the Erlenmeyer flask)

August 5th Wednesday

LCC bacterial expansion and inducing of protein expression:

  1. Add Kan to the LB culture solution (ratio 1:1000).
  2. Add the liquid in the centrifuge tube into the Erlenmeyer flask (the ratio of the LB culture solution to the added liquid in the Erlenmeyer flask is 100:1)
  3. Culture for 4 hours at 37°C (only for Escherichia coli). After 4 hours, the ideal concentration (OD) of the bacterial solution is 0.6-0.8, (Ultraviolet spectrophotometer can be used to determine the bacterial concentration)
  4. Add the protein inducer IPTG (lactose analog) at 16-20 degrees Celsius, and then incubate on a shaker overnight. The concentration of IPTG is 100mM/L

Extract protein & degradation:

  1. Prepare centrifuge tubes, scales, tube racks, and bacteria liquid with IPTG added yesterday
  2. Divide the LCC bacteria liquid into 50ml centrifuge tubes, each centrifuge tube has about 30ml bacteria liquid, use the scale to "peel" to ensure the same weight (there are 15 tubes in total at the end)
  3. Centrifuge the 15 tubes of bacteria liquid, (set-4000 rpm for 30 minutes)
  4. After 30 min, pour out the waste liquid and leave bacteria clumps at the bottom of the centrifuge tube.
  5. Dilute buffer: Tris-hydrochloric acid buffer-1.5Molar diluted 30 times to 50 micromoles per liter
  6. Add 30ml Tris-HCL buffer ph8 in to each centrifuge tube, stir the solution using the tip of the pipette, then resuspend the bacteria clumps.
  7. Add Tris-HCL buffer with bacteria into a beaker, place the beaker in to the ultrasonic disruptor. Time set 15 minutes, set the machine to stop for six seconds after working for every six seconds.
  8. After the Ultrasonic disruption, add the solution back into the centrifuge tubes and centrifuge at 4000 rpm for 30 minutes ((in order to seperate the bacteria remains with the protein)
  9. After the centrifuge is done working, the precipitation are the bacteria remains and the supernatant is the protein wen need. Collect the supernatant into a beaker using a pipette
  10. Put a small piece of PET plastic sheet
  11. Find a same beaker, add the same amount of water as much as the supernatant is. Then add a piece of PET plastic sheet at the same size as the one being put in the supernatant.

Antibiotic-free LB liquid configuration:

  1. Prepare two conical flasks and a measuring cylinder
  2. For 1 Liter, add 5 grams of yeast extract, 10 grams of sodium chloride and 10 grams of peptone into each Erlenmeyer flask.
  3. Add 1000ml of water into each Erlenmeyer flask and shake well
  4. Sterilize these two Erlenmeyer flasks for one hour (Using Kraft paper, tin foil and rubber band cover the mouth of the Erlenmeyer flask) 

August 6th Thursday

  1. Streak pET28a-LCC with 4 petri dishes
  2. Place them on the shaker to store over night

August 7th Friday

  1. Store the streaked petri dish in the 4 degrees Celsius fridge (The laboratory is closed on weekends, and the bacteria are unattended after being picked out and cannot be cultivated according to the plan, so they are stored in the refrigerator first)
  2. Take the four petri dishes (streak, shaker overnight) out of the shaker and put them in the refrigerator at 4 degrees Celsius (forgot to cover the top)

Protein extraction & SDS-PAGE

Using the protein we extracted previously, The protein is divided into 6 centrifuge tubes (the concentration is 1/2n-1 of the first tube)

Equipment:

  • protein loading buffer (6x)
  • 200µl PCR tube x6
  • 5 ml LCC protein
  • Pipette
  • resuspension machine
  • 50 ml of Tris-HCL
  • Add 50 μl LCC protein to the first tube
  • Use a pipette to blow and aspirate to mix the protein loading buffer in the first centrifuge tube with deionized water evenly
  • Resuspend the first centrifuge tube to avoid wall hanging
  • Use a pipette to take 50 microliters of protein and deionized water mixture from the first PCR tube and add it to the second PCR tube, and pipette evenly
  • Resuspend the second centrifuge tube to avoid wall hanging (the protein concentration of the second PCR tube is 1/2 of the first PCR tube)
  • Use a pipette to take 50 microliters of protein and deionized water mixture from the second PCR tube and add it to the third PCR tube, and pipette evenly
  • Resuspend the third centrifuge tube to avoid wall hanging (the protein concentration of the third PCR tube is 1/2 of the second PCR tube, that is, 1/4 of the first PCR tube)
  • Use a pipette to take 50 microliters of protein and deionized water mixture from the third PCR tube and add it to the fourth PCR tube, and pipette evenly
  • Resuspend the fourth centrifuge tube to avoid wall hanging (at this time the protein concentration of the fourth PCR tube is 1/2 of the third PCR tube, that is, 1/8 of the first PCR tube)
  • Use a pipette to take 50 microliters of protein and deionized water mixture from the fourth PCR tube and add it to the fifth PCR tube, and pipette evenly
  • Resuspend the fifth centrifuge tube to avoid wall hanging (at this time, the protein concentration of the fifth PCR tube is 1/2 of the fourth PCR tube, that is, 1/16 of the first PCR tube)
  • Use a pipette to take 50 microliters of protein and deionized water mixture from the fifth PCR tube and add it to the sixth PCR tube, and pipette evenly
  • Resuspend the sixth centrifuge tube to avoid wall hanging (at this time, the protein concentration of the sixth PCR tube is 1/2 of the fifth PCR tube, that is, 1/32 of the first PCR tube)
  • Use a pipette to take 10 microliters of protein loading buffer (6x) into six PCR tubes (six PCR tubes appear electric indigo blue) 

August 10th Monday

Experiment 1: Picking bacteria colony (LCC)
Soak the tip of pipette along with the the colony it picked in a large centrifuge tube (50ml per tube, 4 tubes in total)

Experiment 2: Transforming competent cells

  1. Transfer the pET28a-LCC plasmid into the competent cell
  2. Prepare a box of ice and melt pR6K-pET28a-LCC and p15A-pET28a-LCC in the ice box.
  3. Put the competent cells in the ice box and let it melt, which takes about 1-2m.
  4. After thawing, bring the competent cell to the clean bench and add the plasmid (4ul).
  5. Incubate for 30m on ice.
  6. After incubation, move the tube containing competent cells to the water bath for heat shock for 1minutes at 42 degrees Celsius.
  7. Incubate the competent cells in the ice box for 1-2 minutes.
  8. After incubation, move the competent state to the ultra-clean table and add 500ul Kan-LB liquid.
  9. Insert the tube containing competent cells on the float and put it in the shaker to recover for 1 hour.
  10. Add the Kan-LB liquid on the plate on the petri dish with Kan-LB solid for screening.
  11. Place them in a large constant temperature shaker for cultivation

August 11th Tuesday

Several days ago, we recieved the pR6K and p15A four in four pcr tubes from ShenZhen and each once of them has two tubes. One is with black letters and one is with red letters. We only know that out of these two colors, one stand for plasmid and the other one stands for bacteria, but we couldn’t differ them *Currently, by default, we think black letters are bacteria and red letters are plasmids. The black tube is used for expansion. The scarlet tube is used for transformation (bl21 has grown so it should be a plasmid)

Experiment 1: Picking monoclonal colony

  1. picked the pR6K transformed yesterday and culture in a 50ml large centrifuge tube
  2. Place it in the incubator

Experiment 2: Inducing the expression of LCC

Add IPTG: we have decided to adjust the concentration of IPTG to test the factors affecting the yield of our protein.

  1. Prepare 5 Erlenmeyer flask, with 200ml Kan-LB liquid in each of them
  2. IPTG concentration:
    0
    100:1
    1000:1
    5000:1
    10000:1(Sprinkled in the shaker)
  3. Put them in the shaker overnight
  4. Negative control: BLIS+1000:1 IPTG

Experiment 3: pr6K, p15A expansion

  1. Prepare 50ml centrifuge tube x 50.
  2. Add 5μl to each of the 15ml LB-liquid with Kan-LB medium and Chloramphenicol-LB medium in centrifuge tubes
  3. Culture them on the incubator

Experiment 4: Making Kan and Chl LB solid medium

August 13th Thursday

Experiment 1: pr6k p15a expansion

  1. Add Kan to the LB culture solution (ratio 1:1000). Add the liquid in the centrifuge tube into the Erlenmeyer flask (the ratio of the LB culture solution to the added liquid in the Erlenmeyer flask is 100:1)
  2. Add Chl to the LB culture solution (ratio 1:1000). Add the liquid in the centrifuge tube into the Erlenmeyer flask (the ratio of the LB culture solution to the added liquid in the Erlenmeyer flask is 100:1)
  3. Culture for 4 hours at 37°C (only for Escherichia coli). After 4 hours, the ideal concentration (OD) of the bacterial solution is 0.6-0.8, (Ultraviolet spectrophotometer can be used to determine the bacterial concentration)

Experiment 2: pr6k bacteria preservation

The bl21 pr6k converted the day before yesterday to save the glycerol bacteria into the -80 refrigerator

Experiment 3: LCC protein extraction

  1. Prepare centrifuge tubes, scales, and bacteria liquid induced by IPTG
    • 0iptg
    • 100：1
    • 1000：1
    • 5000：1
    • pr6k 1000：1 (negative control)
  2. Separate the LCC bacteria into centrifuge tubes, use the scale to balance the centrifuge tubes to ensure that they have same weight.
  3. Use the centrifuge, (4000 rpm for 30 minutes)
  4. Get rid of the waste liquid and leave bacteria clumps of p15A-pET28A-LCC at the bottom of the centrifuge tube.
  5. Prepare buffer: Tris-HCL buffer-1.5Molar diluted 30 times to 50 mM/L
  6. Resuspend the bacteria after adding 30ml Tris-HCL buffer
  7. Pour buffer & bacteria into a beaker and place it in the ultrasonic disruptor. Time set 15 minutes, set to stop for six seconds after working for every six seconds
  8. Put the broken bacteria in a centrifuge tube and centrifuge at 4000 rpm for 30 minutes
  9. harvest the supernatant with a beaker

Experiment 4: SDS-PAGE

  1. Clean the required instruments
  2. Dilute the precast gel with tris-HCl buffer
  3. Add extracted LCC supernatent to PCR tubes.
  4. In the order from left to right:
    • 0iptg
    • 100：1 (10µl)
    • 1000：1 (10µl)
    • 5000：1 (10µl)
    • pr6k 1000：1 (negative control,10µl)
    • Protein Marker (6µl)
  5. Add Tris-HCL buffer to each PCR tube
  6. Add the loading buffer of the protein to the PCR tube, the ratio of the loading buffer to the protein solution is 2µl for each.
  7. Centrifuge the liquid on the tube wall to the bottom of the tube in a centrifuge
  8. Put the protein in a water bath and set the temperature to 70 degrees Celsius
  9. Remove the seal under the configured protein gel, fix it on a shelf and close the lid to seal it.
  10. Dilute the SDS buffer with water, to get 1x SDS-PAGE buffer
  11. Pull out the comb and add buffer. (Both positive and negative electrodes are added), the negative electrode is added between the high and low glass,
  12. After setting up the apparatus, add the Protein marker and the prepared sample into the gaps on top of the gel
  13. Turn on the electrophoresis apparatus
  14. Set voltage 100V
  15. When all the protein samples reaches the area between the concentrated gel and the separating gel, switch the voltage to 120V
  16. After the samples reach the bottom of the instrument, turn off the electrophoresis instrument
  17. Take out the gel together with the glass and soak it in water, and pry open the glass with a tool/comb, leaving only the gel
  18. Cut off the strips without samples/markers
  19. Cut off a small corner on the right side of the gel, to distinguish the front and back
  20. Soak the gel in Coomassie Brilliant Blue dye, put it in a horizontal shaker, and shake for one hour
  21. Stop the horizontal shaker and recycle Coomassie Brilliant Blue back into the centrifuge tube
  22. Rinse the protein gel and petri dishes slowly at the tap,
  23. As there are still remaining Coomassie Brilliant Blue left on the gel in the petri dish, add the eluent (the eluent must be over the protein gel) and shake it in a horizontal shaker
  24. Slowly pour the eluent into the pool and rinse the protein gel slowly
  25. Add the new eluent into the petri dish (the eluent must be over surface of the protein gel)
  26. Slowly pour the eluate into the pool after 1 hour.

Experiment 5: LCC protein Degrading PET plastic powder

Add 1g PET powder in to each tube of protein (shown below)

  • 0iptg
  • 100：1 (10µl)
  • 1000：1 (10µl)
  • 5000：1 (10µl)
  • pr6k 1000：1 (negative control,10µl)

Day1

Two tubes of scarlet letters for transformation ➡ Two petri dishes have nothing to grow, and two dishes grow out of other unknown bacteria Conclusion: The scarlet tube is bacteria liquid

Day2

Two tubes of black characters expanded training ➡ did not grow out

Day3

P15a (red letter converted) coated board The pr6k plasmid transformed by the scarlet letter ➡ No result for the red letter pr6k

The previous expansion, transformation, and bacteria preservation were all used wrong

August 14th Friday

Water bath heating

  1. Insert six PCR tubes into the float (protein from yesterday)
  2. Heating electric heating constant temperature water bath, the temperature is set to 72 degrees Celsius, but the highest temperature it reaches is 70 degrees Celsius.
  3. After the electric heating constant temperature water bath is heated to the highest temperature, put the float in and take it out after heating for 10 minutes.

SDS-PAGE

  1. Remove the seal under the configured protein gel, fix it on a shelf and close the lid to seal it.
  2. Dilute the SDS buffer (20ml) with water, the ratio of buffer to water is 1:4
  3. Insert the prefabricated gel, pull out the comb, add a portion of buffer solution first, and check whether the buffer solution in the container leaks
  4. After confirming the tightness, add the buffer solution (fill up) and put it in the tank
  5. Continue to add buffer to the water tank (inside) to reach the maximum volume and continue to overflow. Until the inner side (negative electrode) is full, the outer side (positive electrode) is added to 1/3 to 1/2 and then stop.
  6. Add the LCC protein solution in the PCR tube to the precast gel (the number decreases from small to large)
  7. Finally start the machine (the black wire is connected to the negative pole, the red wire is connected to the positive pole)
  8. When the protein solution reaches the separating gel, adjust the voltage to 120V

Dyeing and bleaching

  1. It finishes working, take out the pre-made gel and the plastic sheet containing the pre-made gel and put them into the metal tray
  2. Use a disposable comb to open the plastic sheet and to take out the preformed gel, and cut off the lower right corner of the preformed gel(for convenience to differ the front and back), and then cut off the part of the concentrated gel
  3. Place the tray on the transfer decoloring shaker, and transfer the precast gel into the petri dish
  4. Add Coomassie Brilliant Blue to the Petri dish until it covers the surface of the gel
  5. Start the transfer decoloring shaker and time for one hour
  6. One hour later, recycle Coomassie Brilliant Blue, and wash the petri dish and the gel simply with water.
  7. Add the eluent and put it on the transfer decolorizing shaker
  8. Start the transfer decoloring shaker and tome for one hour
  9. Change the eluent after one hour, and continue to shake for another hour
  10. Repeat the above steps until the precast gel is completely transparent

August 20th Thursday

  1. bLIS-DH5a primer has been designed
  2. Three tubes of bacteria were picked up from the marked board
  3. Collect yesterday’s bacteria, ultrasonically lyse and extract protein
  4. 1.5L medium plus Kan inoculation bacteria and cultivate overnight
  5. SDS-PAGE, the protein gel after running was decolorized overnight
  6. Add 0.8 g of pet powder to the remaining protein solution into a 72 degree shaker

August 24th Monday

LCC protein extraction

  1. Prepare centrifuge tubes, scales, and bacteria liquid induced by IPTG (this time, we have improved the designing, we uses the µm/L rather than a ratio.
    • 0µM
    • 10µM
    • 50µM
    • 100µM
    • 500µM
    • 1000µM
  2. Separate the LCC bacteria into 6 centrifuge tubes, use the scale to balance the centrifuge tubes to ensure that they have same weight.
  3. centrifuge the 6 tubes, (12000 rpm for 2minutes)
  4. Get rid of the waste liquid and leave bacteria clumps of p15A-pET28A-LCC at the bottom of the centrifuge tube.
  5. Prepare buffer: Tris-HCL buffer-1.5Molar diluted 30 times to 50 mM/L
  6. Resuspend the bacteria after adding 30ml Tris-HCL buffer
  7. Pour buffer & bacteria into a beaker and place it in the ultrasonic disruptor. Time set 15 minutes, set to stop for six seconds after working for every six seconds
  8. Put the broken bacteria in a centrifuge tube and centrifuge at 4000 rpm for 30 minutes
  9. harvest the supernatant with a beaker

SDS-PAGE

  1. Clean the required instruments
  2. Dilute the precast gel with tris-HCl buffer
  3. Add extracted LCC supernatent to PCR tubes.
  4. In the order from left to right:
    • 0µM (10µl)
    • 10µM (10µl)
    • 50µM (10µl)
    • 100µM (10µl)
    • 500µM (10µl)
    • 1000µM (10µl)
    • Marker (6µl)
  5. Add Tris-HCL buffer to each PCR tube
  6. Add the loading buffer of the protein to the PCR tube, the ratio of the loading buffer to the protein solution is 2µl for each.
  7. Centrifuge the liquid on the tube wall to the bottom of the tube in a centrifuge
  8. Put the protein in a water bath and set the temperature to 70 degrees Celsius
  9. Remove the seal under the configured protein gel, fix it on a shelf and close the lid to seal it.
  10. Dilute the SDS buffer with water, to get 1x SDS-PAGE buffer
  11. Pull out the comb and add buffer. (Both positive and negative electrodes are added), the negative electrode is added between the high and low glass,
  12. After setting up the apparatus, add the Protein marker and the prepared sample into the gaps on top of the gel
  13. Turn on the electrophoresis apparatus
  14. Set voltage 100V
  15. When all the protein samples reaches the area between the concentrated gel and the separating gel, switch the voltage to 120V
  16. After the samples reach the bottom of the instrument, turn off the electrophoresis instrument
  17. Take out the gel together with the glass and soak it in water, and pry open the glass with a tool/comb, leaving only the gel
  18. Cut off the strips without samples/markers
  19. Cut off a small corner on the right side of the gel, to distinguish the front and back
  20. Soak the gel in Coomassie Brilliant Blue dye, put it in a horizontal shaker, and shake for one hour
  21. Stop the horizontal shaker and recycle Coomassie Brilliant Blue back into the centrifuge tube
  22. Rinse the protein gel and petri dishes slowly at the tap,
  23. As there are still remaining Coomassie Brilliant Blue left on the gel in the petri dish, add the eluent (the eluent must be over the protein gel) and shake it in a horizontal shaker
  24. Slowly pour the eluent into the pool and rinse the protein gel slowly
  25. Add the new eluent into the petri dish (the eluent must be over surface of the protein gel)
  26. Slowly pour the eluate into the pool after 1 hour.

In conclusion, the experiment we did in Beijing wasn’t as good as expected. Since everyone are high school students who don’t know how to conduct such experiments, it took us weeks to get use to conducting such experiments. The lack of competent equipment is also a reason why we didn’t get the result that we are expecting(the devices are limited, some equipment are severe aging, however, due to the covid-19, this is the only lab we could choose in Beijing.

Shenzhen(Sep 27th - Oct 4th):

Since the results of the experiments in the Laboratory in Beijing didn’t went well, our team negotiated with Link Spider Lab and was authorized to use their Laboratory. Our team had decided to go to Shenzhen and continue our experiments. William went alone during summer break for one week, and four others went again with him during our 1-week long national holiday break.

Sep 26th 2020

Streak BL21(DE3) with pET28a-LCC on three plates

Sep 27th 2020

  1. Make LB liquid 1800 ml, LB solid 300 ml
  2. Make 16 solid LB plates
  3. Transform pET28a-bLIS to BL21(DE3), spread bacteria on solid LB with 50µg/ml Kan
  4. Transform pMVA and pR6K-ptac-GPPS-bLIS to BL21(DE3), spread bacteria on solid LB with 50µg/ml Kan and 35µg/ml Chl
  5. Pre-culture BL21(DE3) with pET28a-LCC into the test tube containing 5ml of LB liquid with 50µg/ml Kan
  6. Pre-culture DH5α with pMVA and pR6K-ptac-GPPS-bLIS into the test tube containing 5ml of LB liquid with 50µg/ml Kan

Sep 28th 2020

  1. Since the BL21(DE3) with pMVA and pR6K-ptac-GPPS-bLIS failed to grow, so we decided to use the same bacteria with same plasmid that Link Spider pre-cultured
  2. Pre-culture the BL21(DE3) with pET28a-bLIS into the test tube containing 5ml of LB liquid with 50µg/ml Kan
  3. Add LB liquid in to 4 Erlenmeyer flasks, each containing 100 ml, add 100µl kan, 75µl Chl in each flask.
  4. Seed culture DH5α with pMVA and pR6K-ptac-GPPS-bLIS in to the LB liquid with 100µl kan, 75µl Chl in a ratio of 1:100 in each of the 4 flasks. (among these four flasks of bacteria, two of them has the od value at around 0.8, one of them has the od value of 1 which is much more than expected and one has the od of 0.4 which is much lower.)
  5. add 2.5µl(25µM) IPTG into each flask of DH5α with pMVA and pR6K-ptac-GPPS-bLIS in 100 ml LB liquid with 100µl kan, 75µl Chl
  6. We group the four flasks DH5α bacteria into two, to ensure each group has a flask of bacteria with od value of 0.8. Add 2.5 ml glucose (20%) in to the flask with od of 0.8 and the flask with od of 1.
  7. Add LB liquid in to 4 Erlenmeyer flasks, each containing 400 ml, add 400µl kan in each flask
  8. Seed culture BL21(DE3) with pET28a-LCC into the LB liquid with 100µl kan in a ratio of 1:100 in each of the 4 flasks
  9. Add 120µl (300µM) IPTG into each flask of BL21(DE3) with pET28a-LCC in 400ml LB liquid with 100µl kan
  10. Place all of the 8 Erlenmeyer flasks in to the shaker(30 Celsius) four of them are 400 ml of BL21(DE3) with pET28a-LCC; four of them are 100 ml of DH5α with pMVA and pR6K-ptac-GPPS-bLIS

Sep 29th 2020

  1. We found that the BL21(DE3) with pET28a-bLIS we pre-cultured yesterday was missing, therefore we pre-culture BL21(DE3) with pET28a-bLIS into four 15mm test tubes containing 5ml of LB liquid with 50µg/ml Kan again.
  2. Divide the four flasks of BL21(DE3) with pET28a-LCC into the test tubes with a measuring range of 50ml, and harvest the bacteria using the centrifuge(8000RPM, 7min)
  3. Make 12%SDS-PAGE gel
  4. Collect the test tubes and eliminate the supernatant, there is one “piece” of BL21(DE3) at the bottom of each test tube. Add 5ml Tris-HCl(20mM) in to each test tube and resuspend the solution with BL21(DE3)
  5. Using the ultrasonic disruptor( power 60, work for 3sec and stop for 6sec. Time set 20min) to break the cell membrane of BL21(DE3) in order to collect the proteins in its cytoplasm.
  6. Use the centrifuge(8000RPM, 7min) again to separate the cell debris with the protein solution, collect the supernatant with new test tubes.
  7. epare 6 pcr tubes, for 4 of the pcr tubes, add 10µl loading buffer of protein and 40µl supernatant of BL21(DE3). For the rest of the 2 pcr tubes, 1 of it add 10µl loading buffer of protein and 40µl of BL21(DE3) with pET28a-LCC that we didn’t Induce protein expression, 1 of it add 10µl of loading buffer of protein and 40µl of DH5α with pMVA and pR6K-ptac-GPPS-bLIS without inducing protein expression.
  8. set up the SDS-PAGE apparatus, add the protein marker and the solution in 6 pcr tubes (1ml protein loading dye and 5ml protein solution) into the gaps at the top of the SDS-PAGE gel. Turn on the gel electrophoresis(100V, 70min)
  9. Add 8ml of His-tag Purification Resin(beads) into 2ml test tube. Using the centrifuge, set 12000RPM for 30sec to collect the beads.
  10. Using pipette to remove 1ml supernatant and add 1ml Tris-HCl. Turn the tube upside down several times to mix the Tris-HCl buffer with the beads. Use the centrifuge with the settings unchanged, repeat this step for 2 times.
  11. Using pipette to remove 1ml supernatant, add 1ml Protein solution in to each tube. Blend them and add the solution from each tube back to the 50ml test tube.
  12. Place the 50ml test tube on to the shaker

Sep 30th 2020

We found the smell of BL21(DE3) with pET28a-bLIS we seed cultured on Sep 28th was weaker comparing to yesterday because the glucose ran out. The two flasks of BL21(DE3) with the OD value of 0.6 functions better.

Protein purification:

  1. Flow through the beads with protein solution in 50ml test tube through the column. Collect the flow through liquid and reserve it in the fridge at 4°C.
  2. Add wash buffer(100mM/L) NaCl into the column, collect it using a 50ml test tube.
  3. Add 5ml Tris-pulldown buffer into the column each time, collect it using a 15ml test tube and repeat this step for 3 times. There are 4 x 5ml purified protein in elution buffer.

Fragrance extraction:

  1. use the two flasks of BL21(DE3) with the OD value of 0.6, add each of them into two 50ml centrifuge tubes, add 4.5ml n-hexane into each tube and shake for 5 min to combine.
  2. Place the flasks under the room temperature, wait until it obviously separates into three different layers and extract the layer(purified linalool) on the top, add the supernatant in to seperated centrifuge tube.

SDS-PAGE

  1. Prepare 2 12% SDS-PAGE gel; set up the SDS-PAGE apparatus
  2. Prepare 7 pcr tubes, in the order from left to right, we have
    • Protein Marker(3ml)
    • Collected flow-through liquid (5ml solution with 1ml loading buffer)
    • Wash buffer (5ml solution with 1ml loading buffer)
    • Elution buffer 1 (5ml solution with 1ml loading buffer)
    • Elution buffer 2 (5ml solution with 1ml loading buffer)
    • Elution buffer 3 (5ml solution with 1ml loading buffer)
    • Elution buffer 4 (5ml solution with 1ml loading buffer)
  3. add Protein loading dye.
  4. Add the marker and samples above in order and turn on the gel electrophoresis(100V, 70min)

Oct 1st 2020

From the result, we see that there are some proteins with the size of 28kd(size of pET28a-LCC) remaining in Flow-through liquid, and wash buffer, and there is another protein bands other than pET28a-LCC in the Elusion buffer. This shows that the protein purification didn’t went perfect, we decided to purify the flow-through buffer to double check if we need to redo it.

  1. Place the flow-through liquid with the beads on a shaker, Combine them for 2 hours.
  2. Flow through flow-through liquid through the column. Collect the flowed through flow-through liquid
  3. Add wash buffer into the column, collect it using a 50ml test tube
  4. Add 5ml Tris-pulldown buffer into the column each time, collect it using a 15ml test tube and repeat this step for 3 times. There are 4 x 5ml purified protein in elution buffer.

Induction of protein expression:

  1. Pre-culture BL21(DE3) with pET28a-LCC into the test tube containing 5ml of LB liquid with 50µg/ml Kan at 10 o’clock in the morning
  2. At 2pm, seed culture BL21(DE3) with pET28a-LCC into the LB liquid with 100µl kan in a ratio of 1:100 in each of the 4 flasks
  3. Divide the four flasks of BL21(DE3) with pET28a-LCC into the test tubes with a measuring range of 50ml, and harvest the bacteria using the centrifuge(8000RPM, 7min)
  4. Make 12%SDS-PAGE gel
  5. Collect the test tubes and eliminate the supernatant, there is one “piece” of BL21(DE3) at the bottom of each test tube. Add 5ml Tris-HCl(20mM) in to each test tube and resuspend the solution with BL21(DE3)
  6. Using the ultrasonic disruptor( power 60, work for 3sec and stop for 6sec. Time set 20min) to break the cell membrane of BL21(DE3) in order to collect the proteins in its cytoplasm.
  7. Use the centrifuge(8000RPM, 7min) again to separate the cell debris with the protein solution, collect the supernatant with new test tubes.
  8. Prepare 6 pcr tubes, for 4 of the pcr tubes, add 10µl loading buffer of protein and 40µl supernatant of BL21(DE3). For the rest of the 2 pcr tubes, 1 of it add 10µl loading buffer of protein and 40µl of BL21(DE3) with pET28a-LCC that we didn’t Induce protein expression, 1 of it add 10µl of loading buffer of protein and 40µl of DH5α with pMVA and pR6K-ptac-GPPS-bLIS without inducing protein expression.
  9. set up the SDS-PAGE apparatus, add the protein marker and the solution in 6 pcr tubes (1ml protein loading dye and 5ml protein solution) into the gaps at the top of the SDS-PAGE gel. Turn on the gel electrophoresis(100V, 70min)
  10. The result is that we do need to redo the protein purification, because there’s another protein that combined with the beads and the wash buffer didn’t washed it down completely.
  11. before we left, we places the the tube containing pET28a-LCC with the beads on the shaker and left them combine overnight.

SDS-PAGE

  1. Prepare 2 12% SDS-PAGE gel; set up the SDS-PAGE apparatus (double sided)
  2. add the protein marker(3ml) and the extracted fragrance(6ml) from 4 separated tubes into the gaps on the top of one of the SDS-PAGE gel.
  3. Prepare 7 pcr tubes, in the order from left to right, we have for one side:
    • Protein Marker(3ml)
    • Collected flow-through liquid (5ml solution with 1ml loading buffer)
    • Wash buffer (5ml solution with 1ml loading buffer)
    • Elution buffer 1 (5ml solution with 1ml loading buffer)
    • Elution buffer 2 (5ml solution with 1ml loading buffer)
    • Elution buffer 3 (5ml solution with 1ml loading buffer)
    • Elution buffer 4 (5ml solution with 1ml loading buffer)
  4. For the other side:
    • Extracted bLIS 1
    • Extracted bLIS 2
    • Extracted bLIS 3
    • Extracted bLIS 4
  5. Add the solutions above in order and turn on the gel electrophoresis(100V, 70min)
  6. After this,After the samples reach the bottom of the instrument, turn off the electrophoresis instrument
  7. Take out the gel together with the glass and simply wash it with water, and pry open the glass with a tool/comb, leaving only the gel
  8. Cut off the strips without samples/markers and the condensed gel part.
  9. Soak the gel in Coomassie Brilliant Blue(Bi Yuntian), put it in a horizontal shaker, and shake for one hour (make sure that Coomassie Brilliant Blue is over the surface of the gel)
  10. Stop the horizontal shaker and recycle Coomassie Brilliant Blue back into the centrifuge tube
  11. Rinse the gel and petri dishes gently
  12. As there are still remaining Coomassie Brilliant Blue left on the gel in the petri dish, add water and shake it in a horizontal shaker over night.

Oct 2nd 2020

  1. we found the four BL21(DE3) with pET28a-bLIS in 15mm test tubes we pre-cultured on Sep29th at the clean desk
  2. Use the BL21(DE3) with pET28a-LCC we extracted yesterday to redo the protein purification:
  3. Flow through the beads with protein solution in 50ml test tube through the column. Collect the flow through liquid and reserve it in the fridge at 4°C.
  4. Add wash buffer(300mM) into the column, collect it using a 50ml test tube.
  5. Add 5ml Tris-pulldown buffer into the column each time, collect it using a 15ml test tube and repeat this step for 3 times. There are 4 x 5ml purified protein in elution buffer.

Using the microplate reader

  1. Preparation of protein standard: completely dissolve protein standard at room temperature, take 20 µl of 5mg/ml BSA protein standard solution for use. Dilute the PBS solution to 100 µl to make the final concentration 1.0 mg/ml.
  2. Prepare the BSA standard measurement solution according to the following table:
    Number 0 1 2 3 4 5 6 7 8
    1 mg/ml BSA standard solution (μl) 5 mg/ml BSA standard solution (μl)
    BSA standard solution (µl) 0 0.5 2.5 5.0 10 15 20 6 8
    PBS solution (μl) 20 19.5 17.5 15 10 5 0 14 12
    BSA final concentration μg/ml 0 25 125 250 500 750 1000 1500 2000
    total capacity µl 20 µl
  3. Add sample of pET28A-LCC (1µl, 2µl, 5µl) into three microtiter plate and make up to 20 µl with PBS
  4. Add 200 μl of BCA working solution to the microplate, mix well, and place at 37°C for 30 minutes;
  5. Measure the absorbance at 562 nm and record the reading; use the absorbance of the sample without BSA as a blank control.
  6. Using A562 as the ordinate and BSA content as the abscissa, draw a standard curve to calculate the protein concentration in the sample. If the obtained protein concentration is not within the range of the standard curve, please dilute the sample and measure again.

Degradation Experiment:

From the article “An engineered PET depolymerase to break down and recycle plastic bottles” the research team uses the LCC with the density of 0.69mM/ml, we use it as a standard density
0.69µM x 3, 2µM x 1, 3µM x 1

Oct 3rd 2020

LCC（50mM NaCl) purification:

  1. Flow through the beads with protein solution in 50ml test tube through the column. Collect the flow through liquid and reserve it in the fridge at 4°C.
  2. Add 5ml Tris-pulldown buffer into the column each time, collect it using a 15ml test tube and repeat this step for 3 times. There are 4 x 5ml purified protein in elution buffer.
  3. Since the purification today was failed, we then do a SDS-PAGE for yesterday’s purification (same steps as before) and we found out that elusion2 and elusion5 was combined perfectly. Which means that 300mM/L NaCl for wash buffer works better than 100mM/L for pET29A-LCC.
  4. Redo SDS-PAGE for bLIS (the same steps with Oct.1st.) since the concentrations between them were so different. After the gel was dyed, we decolorized it using water.

Concentrate Protein:

  1. prepare a 50ml protein concentrator, add some Tris-HCL and shake it gently to clean the remained protein from the last experiment
  2. Add the protein in to the purified protein solution into the concentrator
  3. Use another test tube and add water to balance their weight.
  4. Put them in the centrifuge (8000rpm, 30min)
  5. Get rid of the waste liquid at the bottom of the protein concentrator, add protein until the upper part is full.
  6. Repeat step 4 and 5 for 3 times, until we have concentrated all of our protein.

Degradation Experiment:

From the article “An engineered PET depolymerase to break down and recycle plastic bottles” the research team uses the LCC with the density of 0.69mM/ml, we use it as a standard density, and the below are the gradients of our protein concentration. (LCC volume and Tris-HCL buffer volume.)

Since we have 600µl of purified concentrated protein, we could

concentration (uM/ml) 0.25 0.5 5 10 15 20
volume (ul) of LCC 1.99 3.98 39.8 79.6 119.4 159.2
volume (ul) of Tris 998.01 996.02 960.2 920.4 880.6 840.8

Oct 4th 2020

Eventually, for the last day of the experiment, our plan was completed and we decided to do one last SDS-PAGE to show our best result, and is also a comprehensive one.

  • Marker 3µl
  • Supernatant 5µ
  • sediment 3µ
  • Flow-through liquid 5µl
  • Wash buffer 5µl
  • E1 5µl
  • E2 5µl
  • E3 5µl
  • E4 5µl
  • negetive control (bLIS) 3µl
  • negative control of negetive control bLIS 3µl

Then, we have sent the test tubes containing the our protein solution (0.25µM/ml, 0.5µM/ml 0.69µM/ml, 2µM/ml, 3µM/ml, 5µM/ml, 10µM/ml, 20µM/ml) to do GC

Almost all of the results of our experiment comes out in Shenzhen (degradation experiment, Enzyme activity analysis, fragrance extraction, protein purification, protein concentration and etc.), and it generally met our expectation. Although our experiment in Beijing isn’t productive, but we had a lot of practice and learn lessons in failure again and again; this is also a reason why we get everything done within one week.