Team:Worldshaper-Shanghai/Protocol

Protocol

Test for part’s function

Plan A

We build two plasmids: the first is pHucO-mCherry, and the second is HucR-pHucO-mCherry. We transfer these plasmids in the E. coli and test the function of pHucO and HucR. Then, we use uric acid to induce the fall off of HucR and wait for the rehabilitation of pHucO’s function.

Plasmid Extraction

  1. Add 1 ml overnight bacteria liquid to an EP tube. Centrifuge at 12,000 rpm for 1 min. Remove the supernatant.
  2. Add 100 μl Solution 1. Resuspend the bacteria with pipette.
    Note: Be sure the bacteria are resuspended completely.
  3. Add 200 μl Solution 2. Mix by gently inverting the tube for 4-6 times. The solution becomes transparent.
  4. Add 150 μl Solution 3. Mix by gently inverting the tube for 8-10 times. White flocculent precipitate appears. Put the tube in ice bath for 5min.
  5. Centrifuge at 12,000 rpm for 10 min. Transfer the supernatant to a new EP tube.
  6. Add 1×isopropanol. Centrifuge at 12,000 rpm for 15 min. Remove the supernatant. Dry the precipitate by opening the tube till it becomes colorless.
    Note: Be sure the supernatant is removed completely.
  7. Add 1 ml 75% ethanol. Centrifuge at 12,000 rpm for 1 min. Remove the supernatant.
  8. Add 20 μl TE solution (20 μg/ml RNase)
Solution 1 (pH = 8.0) 10mM EDTA
25mM Tris-HCl
Solution 2 0.2M NaOH
1% SDS
Solution3 3M Potassium Acetate
2M Acetic Acid

Electrophoresis Gel

  1. Add 0.64 g Agarose to a flask.
  2. Add 80 ml 1×TAE solution to the flask.
  3. Heat the solution till bubbles appear. Mix the solution. Repeat the process 2-3 times till the solution becomes transparent.
  4. Add 8 μl DuRed to the solution after it is cooled to room temperature. Mix the solution.

Transformation

  1. Add 500 μl host cell in a receptive state and 1 μl plasmid in an EP tube.
  2. Put the tube in ice bath for 30 min.
  3. Put the tube in 42°C water bath for 90 s.
  4. Put the tube in ice bath for 3 min.
  5. Shake the tube at 180 rpm under 37°C for 1 h.
  6. Centrifuge at 12,000 rpm for 1min. Remove most of the supernatant and keep about 50 μl in the tube.
  7. Resuspend the bacteria with pipette. Coat 50 μl bacteria on culture medium plate. Incubate the plate at 37°C overnight.

PCR

System:

Reagent Volume (μl)
dH2O 14.4
dNT 2.0
10×Buffer 2.0
5’ primer 0.2
3’ primer 0.2
Enzyme 1.0
Template 0.2

Procedure:

Step Temperature (°C) Time
Initial Denaturation 94 30 sec
30 Cycles 94 15-30sec
45-68 15-60sec
68 1min per kb
Final Extension 68 5min

Restriction Enzyme Digestion

Time:

For the digestion of target fragment, the length of the digestion is 4 h. For the digestion of plasmid, the digestion will be conducted overnight.

System for digestion of vector:

Reagent Volume (ml)
pHucO-mCherry-pS1BC3 Plasmid 5.0
10×M Buffer 2.5
EcoRI 1.0
XbaI/td> 1.0
ddH2O 15.5

System for digestion of target fragment:

Reagent Volume (ml)
pc+HucR-histag-pSB1C3 Plasmid 10.0
10×H Buffer 2.5
EcoRI 1.0
SpeI 1.0
ddH2O 10.5

Procedure:

  1. Determine the enzyme digestion system. Turn the temperature setup of the water bath to 37°C.
  2. Prepare the ingredients. Unfreeze the enzyme digestion substrates and 10×enzyme digestion buffer on ice. Unfreeze and add the enzyme after other kinds of reagents are added.
  3. Add the ingredients to a 1.5ml EP tube according to the system set below. The order is: ddH2O, 10× enzyme digestion buffer, enzyme digestion substrate, enzyme.
  4. 4. Use pippet with appropriate measuring range to blend the solution. Put the EP tube on the buoy and put it in the water bath with the temperature of 37°C.

Connection Reaction

System:

Reagent Volume (ml)
Target Fragment 7.0
Vector 1.0
T4 Ligation Buffer 1.0
T4 Ligase 1.0

Notes ： To achieve a most efficient connection, try best to control the amount of the reaction system within 10ul.

Procedure:

  1. Take out Ligation Buffer from -20°C then melt it on ice
  2. Prepare the DNA solution which will be used for connection: add enzyme digested target fragment and recycled product of the vector in proportion (usually the molar ratio of target fragment and the vector is ranged from 4:1 – 10:1)
  3. Add enzyme digested vector and fragment in the following proportion, react at 16°C for 4h or overnight

LB medium and LB plate

System for LB medium (pH 7.0):

Reagent Quantity
Tryptone 10g
Yeast Extract 5g
NaCl 10g
H2O Makes up to 1L
System for LB plate:
Reagent Quantity
Tryptone 10g
Yeast Extract 5g
NaCl 10g
Agarose 15g
H2O Makes up to 1L

Plan B

We use EMSA to test the combination of HucR and pHucO.

The biotin-labled pHucO probe was synthesised and modified by Beijing TsingKe Biotech , Co., Ltd. The HucR-mCherry fusion protein was purified as following：

protein purification（preparation for the next experiment depicted）

Pre-experiment for the protein purification

  1. configurate 5X TBE :

    • 10.8g Tris base
    • 5.5g boric acid
    • 4ml 0.5M EDTA

    Add all those compounds above into 200ml distilled water

  2. configurate different concentrations of wash buffer (NT Buffer+ imidazole)

Purifying the HucR-mcherry fusion protein

  1. Add 10 ml NT Buffer in to 2ml Ni+ column, dispose the filtrate, then add certain amount of NT Buffer in order to keep the balance
  2. Pipette 1 ml HucR-mcherry fusion protein into 4, 2ml centrifuge tubes and 3, 1.5 ml centrifuge tubes
  3. Centrifuge those 7 tubes, sets under 10min, 25°C, 15000 rpm ( the purpose of this step is to separate the remaining solid in the protein, mainly is to assure its purity )
  4. Pipette 900 ul the supernatant from the centrifuged fusion protein, add it into the Ni+ column
  5. Double check whether the mixture is perfectly mixed, then put it in a ferris wheel, sets under 7.5r/rpm ( in order to let the roughly purified fusion protein connect tightly to the Ni+ column, preventing the sedimentation )
  6. Settle the mixture naturally, wait for the gravitational force to separate the target fusion protein (HucR-mcherry) and those useless protein( mainly those remaining proteins provided by the E.coli itself)

The upper level remains the initially purified fusion protein, the remaining part accumulates those “useless” proteins

Wash of the fusion protein

  1. The first wash: add 7ml ,25Mm wash buffer (1 ml imidazole+19ml NT buffer) into the fusion protein

    The second wash: add 10ml 50mM wash buffer (2 ml imidazole+18 ml NT buffer)

    The third wash: add 10ml 75mM wash buffer（3 ml imidazole+17 ml NT buffer）

  2. the last process for the ultimately purified fusion protein: add 1 ml 500mM Ni+ column, which pH=8, ( 50mm Tris,150mm Nacl,500mM imidazole）, then put it into the shaker for 5 min

EMSA protocol

Figure 1 Protocol
Figure 2 Protocol
Figure 3 Protocol
Figure 4 Protocol
Figure 5 Protocol

Observe EMSA gel

  1. Add 15ml sealing fluid in a vessel. Add crosslinking nylon membrane (containing sample) in and seal for 30min.
  2. Add 7.5ml Streptavidin-HRP Conjugate in 15ml sealing fluid (1:2000 diluted) for preparation.
  3. Remove the sealing fluid in the vessel. Add the 15ml sealing fluid with Streptavidin-HRP Conjugate. Shake the vessel for 30min.
  4. Rinse the nylon membrane with scrubbing solution for 1min.
  5. Remove the scrubbing solution. Add 15-20ml scrubbing solution in the vessel. Shake it for 5min. Repeat the step for three times.
  6. Take out the nylon membrane and remove excessive water with absorbent paper. Turn the facet with sample upward immediately and place it in a clean vessel horizontally.
  7. Add ECL solution on the surface of the nylon membrane till it is completely covered. Wait for 5min.
  8. Take out the nylon membrane and remove excessive water with absorbent paper. Put the nylon membrane amid two euphotic membranes and develop it.

Test for Project Design

Combination between pHucO fragment and HucR-mcherry fusion protein

  1. Mark on the cellulose acetate membrane by pencil, in order to assure the pHucO’s positions, each membrane contains 7-8 marks
  2. Pipette 5μl, 10μM pHucO DNA fragment, make sure it is on the position marked by pencil previously (when the membrane is dried out completely there should be nothing left on it)
  3. Put the membrane with the DNA fragment into UV crosslinking instrument, set under: 120mJ/cm2, 1min (the purpose of this step is to double check whether the binding between the DNA fragment and the membrane is strong enough in order to prevent the possible flushing during the elution)
  4. Place the membrane in a petri dish, add PBS solution for elution, then put the dish onto a shaker for 2min (this step mainly serves to remove the DNA fragment which failed to bind with the membrane)
  5. Repeat the elution according to the steps described in 4
  6. Cut up 24 pieces of membrane each containing only one pHucO fragment and put them into a 24 well-plate
  7. Pipette 50 μl，20μg/ml HucR-mcherry into each 24 wells containing the membrane, shake it for 15 mins

Addition of the UA

  1. Prepare different concentrations of UA, 0μM/ 100μM/ 200μM/ 300μM/ 400μM/ 450μM/ 500μM/ 600μM/ 700μM/ 800μM/ 900μM/ 1000μM respectively

  2. 900μM UA: 900μl, 1000μM UA solution+100μl PBS

    800μM UA: 800μl, 1000μM UA solution+200μl PBS

    100μM UA: 100μl, 1000μM UA solution+900μl PBS

  3. Pipette 500μl of UA solution with different concentrations into the 24 well- plate in the following order:

    UA:0μM (500μl) UA:100μM (500μl) UA:200μM (500μl) UA:300μM (500μl) UA:400μM (500μl) UA:450μM (500μl)
               
    UA:500μM (500μl) UA:600μM (500μl) UA:700μM (500μl) UA:800μM (500μl) UA:900μM (500μl) UA:1000μM (500μl)

    NOTE: the concentration of the fusion protein and the DNA fragment are the same for all 24 wells, 20 μg/ml and 10μM respectively (the blanks which contain the same color represents that all the protein, DNA and UA solution added there are the same)

  4. Remove all liquid, observe and photograph the membranes under spectrometer, Excitation / Emission: 540nm/620nm.