Team:CUHKSZ/pages/protocol

EthaNO - CUHKSZ

Protocol

Culture of Escherichia coli and growth curve

Materials:
Equipments:
  1. E. coli colonies on solid medium
  2. Sterile Erlenmeyer flasks with caps
  3. Pipettes and pipette tips
  4. Cuvettes
  5. A spectrophotometer
Consumables:
  1. E. coli colonies on solid medium
  2. Luria-Bertani (LB) broth
Protocol:
E. coli culture
  1. Using aseptic technique, carefully transfer a 10-ml aliquot of LB broth into a capped, sterile Erylenmeyer flask.
  2. With a sterile pipette tip, touch a single, well isolated colony on the solid medium plate. Resuspend the picked colony in the liquid medium.
  3. Replace the cap on the Erlenmeyer flask and incubate the flask in a 37 °C incubator with aeration 180 r.p.m.) overnight.
Measure the growth curve
  1. Vortex overnight culture to resuspend the cells. Remove 30 μl of overnight culture into 30 ml LB broth to inuculate. Culture the cells under 37 °C with aeration 180 r.p.m.
  2. Each time, swirl flask to evenly distribute the cells and aliquot 2 ml of the culture into a cuvette. Use 2 ml LB broth in another cuvette as blank control. Mesure the optical density (560 nm) using a spectrophotometer.
  3. Before entering log phase, repeat step 2 every hour. When OD reaches about 0.8, measure the optical density every 30 minutes.

Storage of Escherichia coli frozen stocks in glycerol

Materials:
Equipment:
  1. Pipettes and pipette tips
  2. Centrifugator
  3. 1.5 ml microfuges
  4. -80 °C freezer
Consumables:
  1. E. coli liquid culture
  2. Luria-Bertani (LB) broth, sterile
  3. 30 % (v/v) glycerol
Protocol:
  1. From E. coli liquid culture, alliquot 1.2 ml into the microfuge. Centrifuge the cells at 3000 rpm for 5 minutes. Carefully discard the supernatant. Add another 1.2 ml culture and centrifuge. Repeat 3 times.
  2. Resuspend the cell pellets with 0.5ml LB broth. Add 0.5ml of 30% (v/v) glycerol, and vortex to mix well.
  3. Label the vial and cap with appropriate strain names and other relevant information and freeze immediately in a -80°C freezer.

3. Culture of acetobacter pasteurianus

Materials:
Equipment:
  1. Erlenmeyer flasks with cap
  2. Pipettes and pipette tips
Consumables:
  1. Tryptone
  2. Yeast extract
  3. Glucose
  4. Anhydrous ethanol
Protocol:
Deploy culture media:
  1. 10 g/L tryptone, 5 g/L yeast extract, 20 g/L glucose, heat until everything dissolves. Cover the flasks with membrane. Autoclave under 115 °C for 15 minutes.
Acetobacter pasteurianus culture:
  1. Use a sterile pipette tip to transfer a single colony from the solid medium plate into 25 ml culture medium. Culture in a 30 °C incubator with areation of 180 r.p.m. overnight.
  2. Prepare four sterile Erlenmeyer flasks, add 50 ml of culture medium to each flask. Add 1 ml, 2 ml and 3 ml anhydrous ethanol to three flasks respectively and label the flasks.
  3. Add 50 μl overnight cell culture into each flask. Culture for at least 24 hours to observe the growth situation.

Transformation of Escherichia coli by heat shock

Materials:
Equipment:
  1. Pipette and pipette tips
  2. Eppendorf tubes
  3. Centrifuge
  4. Flasks
  5. Heat bath
Consumables:
  1. Reconstructed plasmid, 1ng/μl in TE buffer.
  2. ddHO
  3. 0.1 M CaCl
  4. Luria-Bertani broth media
  5. LB plates with ampicillin
Protocol:
Competent cells preparation:
  1. Culture the E. coli cells in 50 ml LB broth to mid-log phase. The mid-log phase generally has an absorption value of OD between 0.6 and 1.5.
  2. Place about 40 ml of bacterial culture in a sterile 50 ml centrifuge tube and spin at 4000 r.p.m. for 5 minutes at 4 °C in a Eppendorf table-top centrifuge.

The following procedures should be performed on ice for maximal transformation rate.

  1. Carefully remove the supernatant and resuspend the cells with 4 ml ice-cold 0.1 M CaCl. Incubate the bacterial suspension on ice for 30 minutes.
  2. Centrifuge at 4000 r.p.m. for 5 minutes at 4 °C. Remove the supernatant. Add 0.1 ml ice-cold 0.1 M CaCl to resuspend the pellet. Keep the tube on ice.
Heat shock induction:
  1. Add 100 μl bacteria solution and 1 μl plasmid solution to a 1.5 ml pre-chilled microfuge tube. Incubate the tube on ice for 20 minutes.
  2. Place the tube in water bath at 42 °C for 45 seconds. Immediately place the tube on ice for at least 3 minutes.
  3. Add 1 ml room temperature LB liquid media to each tube. Incubate the tube under 225 r.p.m., 37 °C to recover the bacteria.

Ethanol decomposition

Materials:
Equipment:
  1. Pipettes and pipette tips
  2. Erylenmeyer flasks
  3. Microfuge tubes
Consumables:
  1. Luria-Bertani broth media
  2. Acetobacter culture media
  3. ddHO
  4. 50 mg/ml IPTG
Protocols:

  1. Culture the E. coli BL-21, E.coli BL-21 with plasmid and acetobacter, each in 500 ml culture media. Store them under 4 °C.

  2. Before use E. coli BL-21 (plasmid) culture, recover the cells in 37°C incubator for 2 hours, add 50 mg/ml IPTG in a ratio of 1/10, incubate for 30 minutes.

  3. Deploy each experiment set according to the table:

    酒精浓度 0% 2% 4% 6% 8%

    实验组别
    无菌水10mL 1 2 3 4 5 BL-21 10mL 6 7 8 9 10 BL-21(plasmid）10mL 11 12 13 14 15 BL-21 5mL+巴氏醋杆菌 5mL 16 17 18 19 20 BL-21(plasmid） 5mL+巴氏醋杆菌 5mL 21 22 23 24 25

  4. Take 1 ml of the mixture and store as blank control. Culture the remaining bacteria for 14 hours.

  5. Use Ethanol Colorimetric/Fluorometric Assay Kit to measure the alcohol concentration.

Testing proper concentration range of PVA/SA cross-linking beads

Materials

Consumables:

Poly vinyl alcohol, 1788

Sodium alginate

Boric acid

CaCl2 Analytical Titrant, 0.5M

 

Equipment:

100 ml cylinder

Analytical balance

Experiment containers

100 ml Beakers

Protocol

1.     PVA and Sodium alginate with different concentrations are mixed in beakers as shown in chart 1. The mixture is then put in 80% for 4h to get fully dissolved.

2.     The solution above is added to 10 ml immobilizing (boric acid + 2/3/4% CaCl2) solutions in the experiment containers with 10 ul pipette, yielding 20 beads and kept in 4°C refrigerator forming for 24h.

3.     Heat dry the liquid up after the bead were collected from the immobilizing solution. Collecting and observing the bead properties (as unshaped, flat and formed), and the formed ones should be retained in 4°C water for further tests.

Testing ethanol degradation rate of PVA/SA cross-linking beads (with E. coli and A.A.B.)

Materials

Consumables:

Poly vinyl alcohol, 1788

Sodium alginate

Boric acid

CaCl2 Analytical Titrant, 0.5M

Ethanol

Equipment:

50 ml cylinder

Shaker

100 ml Beakers

Ethanol meter

Protocol

4.     8% PVA and 2.5% Sodium alginate are mixed in beakers as shown in chart 1. The mixture is then put in 80% for 4h to get fully dissolved.

 

5.     The solution above is added to 10 ml immobilizing (boric acid + 3% CaCl2+1ml E.coli and ACE) solutions in the experiment containers with 10 ul pipette, yielding 20 beads and kept in 25-degree shaker forming for 24h.

 

6.     The bead were collected from the immobilizing solution. The beads are then washed up with SPSS. The beads is then put into 20ml 5% ethanol solution. Record the concentration of ethanol with ethanol meter every 24 hours.

 

Testing bacteria permeability of PVA/SA cross-linking beads

Materials

Consumable:

Poly vinyl alcohol, 1788

Sodium alginate

Boric acid

LB broth

Acetobacter pasteurianus cultural media

CaCl2 Analytical Titrant, 0.5M

Equipment:

Spectrometer

Protocol:

1.     Take standard ingredient (PVA 8%, SA 3%, CaCl2 3%, 10% bacteria mixture) of beads from protocol 1.

2.     Cultivating ‘standard’ beads into LB broth and A. pasteurianus cultural media for 24 h. Measuring optical density of 560 mm and 600nm, corresponding to E. coli and Acetobacter, to see whether the bacteria cells would leak out of beads and grow.

 

Using Plackett-Burman Design to get decisive variables for the beads’ ethanol degradation

Materials

Consumable:

Poly vinyl alcohol, 1788

Sodium alginate

Boric acid

Sodium boride

CaCl2 Analytical Titrant, 0.5M

Equipment:

Ethanol meter

Protocol

1.     The levels are designed as shown in table 1:

 

Level

PVA (%)

SA (%)

CaCl2 (%)

Time (h)

pH

Bac. Inoculation (%)

-1

7

2

2

24

6

10

0

8

3

3

48

7

15

1

9

4

4

72

8

20

           

            And the beads are prepared as shown in following table 2, each with 0.1 ml volume.

 

2.     50 beads are then added into 4% alcohol in the container. The alcohol concentration is then measured with ethanol meter and put into constant temperature shaker, 37 °C to cultivate for alcohol degradation. After cultivation, the alcohol concentration is again measured. The data is then collected for Minitab’s Placket-Burman analysis.

 

Group

Time

PVA

SA

CaCl2

pH

Bac. inoculation

1

-1

1

1

-1

1

-1

2

-1

-1

1

1

1

-1

3

-1

-1

-1

1

1

1

4

1

1

1

-1

1

1

5

-1

1

-1

-1

-1

1

6

1

1

-1

1

-1

-1

7

1

-1

1

-1

-1

-1

8

1

1

-1

1

1

-1

9

-1

1

1

1

-1

1

10

1

-1

-1

-1

1

1

11

-1

-1

-1

-1

-1

-1

12

1

-1

1

1

-1

1

13

0

0

0

0

0

0

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