Team:KEYSTONE/Protocol

HOME
JUDGING FORM Judging form
SAFETY Safety

Antibiotic-free LB liquid

  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)

Make competent cells

Materials:

  • Centrifuge tube
  • Jars
  • Calcium chloride
  • Glycerin
  • Deionized water

Procedure:

  1. Prepare calcium chloride solution
  2. Use a pipette to add 4 ml of glycerol to a 5 ml centrifuge tube on the clean bench.
  3. Add calcium chloride and deionized water to a jar at a concentration of 0.1M/L. Note: The relative molecular mass is 110.99, so for example, 1.1 grams should be added to 100 ml of deionized water.
  4. Prepare the clump
  5. Prepare glycerin
  6. Resuspended

Plasmid amplification

  1. Buy plasmid: powder + bacterial solution (Top10/DH5alpha/Transalpha amplified strain)
  2. Bacteria enlarge culture
  3. Extract plasmid (miniprep)
  4. DNA Gel electrophoresis
  5. Bacteria preservation (Top10) -80˚C refrigerator, add glycerin (to prevent cells from being punctured by ice crystals & freezing)

Plasmid Extraction

Materials:

  • Bacteria
  • Centrifuge
  • BufferS1
  • BufferS2
  • BufferS3
  • BufferW1
  • BufferW2
  • Elution buffer
  • Column

Procedure:

  1. 1-4ml of bacteria liquid, centrifuge at 12000rpm for 1 minute to separate the bacteria and discard the waste liquid
  2. Add 250µl Buffer S1 to the bacteria and shake or pipette
  3. 250µl of S2 solution was added and gently shaken to mix
  4. Add 350µl S3 solution and gently invert 6-8 times
  5. Centrifuge at 12000rpm for 10 minutes
  6. Transfer the centrifuged supernatant to the column
  7. Add 500µl bufferW1 and centrifuge for 1 minute
  8. Aspirate waste
  9. Add 700µl W2 and centrifuge for 1 minute
  10. Change a lower ep tube
  11. Add 60μl of elution buffer or deionized water and centrifuge for 1 minute (If there are multiple columns, add them all into one ep tube to increase the concentration)Noted: Store in -20 degrees refrigerator

Protein induced expression

Materials
Bacteria (not induced)
Shaker
Centrifuge tube
Clean Desk

Culture:

  1. Use the tip of the pipette to pick a colony in the Petri dish.
  2. Put the pipette tip with bacteria into a centrifuge tube with 10ml-20ml Kan medium and pre-culture overnight on a shaker
  3. 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)
  4. 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)
  5. 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
  6. Extract Protein
  7. SDS-Page

Protein Extraction

Materials:

  • Centrifuge
  • Buffer
  • Tris-HCl buffer
  • Ultrasonic crusher
  1. Prepare centrifuge tubes, scales, tube racks, and bacteria liquid induced by IPTG
  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 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 with 30ml Tris-HCL buffer
  7. Buffer & bacteria are put into ultrasonic disintegration. 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. Collect 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. Add 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
    Set voltage 100V
    When all the protein samples reaches the area between the concentrated gel and the separating gel, switch the voltage to 120V
  13. After the samples reach the bottom of the instrument, turn off the electrophoresis instrument
  14. Take out the glue together with the glass and soak it in water, and pry open the glass with a tool/comb, leaving only the gel
  15. Cut off the strips without samples/markers
  16. Cut off a small corner on the right side of the glue, to distinguish the front and back
  17. Soak the gel in Coomassie Brilliant Blue dye, put it in a horizontal shaker, and shake for one hour
  18. Stop the horizontal shaker and recycle Coomassie Brilliant Blue back into the centrifuge tube
  19. Rinse the protein gel and petri dishes slowly at the tap,
  20. 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 to 5:20 in a horizontal shaker
  21. Slowly pour the eluent into the pool at 5:20 and rinse the protein glue slowly
  22. Add the new eluent into the petri dish (the eluent must be under the protein gel)
  23. Slowly pour the eluate into the pool in the morning of the next day and rinse the protein gel slowly

Protein purification

  1. Add beads into a 50ml test tube containing protein supernatant, shake it using the shaker overnight to fully combine them.
  2. Flow through the protein combined with beads using a column, and use a 50ml test tube to collect the flow through.
  3. Add the prepared wash buffer to the column, and collect it below with a 50ml test tube
  4. Add His-pull down, to the filter test tube, use 15ml test tube to collect, each 5ml is a group to collect four groups

Yeast culture solution

Materials:

BMGY (growth):

  • Yeast extract 1%
  • Peptone 2%
  • YNB Amino acid-free yeast nitrogen source 1.34%
  • Biotin 4x10^-5% (0.00001%, 0.1μg per liter)
  • Glycerol 1%
  • K3PO4 potassium phosphate 1.36% medium concentration 100mM

BMMY (induction):

  • Yeast extract 1%
  • Peptone 2%
  • YNB Amino acid-free yeast nitrogen source 1.34%
  • Biotin 4x10^-5% (0.00001%, 0.1μg per liter)
  • K3PO4 potassium phosphate 1.36% medium concentration 100mM
  • Methanol 3% *LD=1g/kg

Configure culture medium

  1. Prepare the materials in proportion and add them to the conical flask.
  2. add water
  3. Autoclave sterilization

DNA Gel Electrophoresis

Materials:

  • 50xTAE
  • Water
  • Agarose
  • DNA loading dye
  • Gel Electrophoresis apparatus
  • Microwave
  • Electrophoresis
  • DNA Marker
  • DNA Loading buffer

Procedure:

  1. prepare gel for DNA Gel Electrophoresis
  2. Glue: TAE+Agarose (agarose) 2% microwave heating for two minutes to dissolve
  3. Add DNA dye (pink), the ratio is 1/10000 *Note: a layer of PE gloves on rubber gloves to prevent skin cancer
  4. Put the comb on the leftmost side of the tray (if there are more samples, put two)* Leave a little gap with the bottom of the tray
  5. Pour the glue into the tray and wait for it to solidify completely
  6. Plasmid sample and loading dye are mixed according to the ratio marked on the dye(1:5)
  7. Add TAE into the electrophoresis tank (no gel tray)
  8. Take out the comb*Do not shake
  9. Put the transparent tray into the electrophoresis tank, with the gaps side facing the negative electrode
  10. Inject DNA Marker into the most side hole of the tray *Do not poke glue to avoid overflow
  11. Inject sample into other holes
  12. Close the electrophoresis tank and start the electrophoresis device
  13. Put the gel in the gel imager to read

Transform competent cells

Materials:

  • A box of ice cubes
  • One tube of competent cells
  • 1ul plasmid
  • 500ul of anti-LB medium
  • Several solid petri dishes
  1. Prepare a box of ice; Put the competent state in the ice box and let it melt, which takes about 1-2m.
  2. After thawing, move the competent state to the ultra-clean table, and add the plasmid (1ul).
  3. Incubate for 30m on ice.
  4. After incubation, move the competent tube to a hot water pot for heat shock for 1m. 42 degrees
    Incubate 1-2m on ice.
  5. After incubation, move the competent state to the ultra-clean table, and add 500ul anti-LB medium.
  6. Insert the competent tube on the float and put it in the shaker to recover for 1 hour.

Fragrance extraction (for linalool):

  1. Add the desired bacteria liquid (BL21(DE3)) 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 to reserve

Degradation Experiment

Materials:

  • PET powder
  • Tris-HCL buffer
  • potassium phosphate buffer
  • 100ml erlenmeyer flasks

Procedure:

  • Prepare 1 ml of a 0.69 µM solution of purified protein in 20 mM Tris-HCl（PH8）, add 300 mM NaCl
  • Prepare a 100ml erlenmeyer flask. Add 49 ml of protein-specific buffer. Choice of protein-specific buffer has been done according to scientific literature: 100 mM potassium phosphate buffer pH 8 is for LCC41.
  • Add 200mg PET powder into the 100ml erlenmeyer flasks and place it on the incubator at desired temperature(70 degrees of Celsius) under agitation at 170 rpm.

Microplate reader

Microplate determination procedure: (working range 20-2000 μg/ml)

  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
  3. Add an appropriate volume of the sample to be tested into the 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;
    Note: It can also be placed at room temperature for 2 hours, or at 60°C for 30 minutes. When the BCA method is used to determine the protein concentration, the color will deepen over time, and the color reaction will be accelerated due to the increase in temperature. If the concentration is lower, it is suitable to incubate at a higher temperature or extend the incubation time appropriately.
  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.

Protein Concentration

  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.