Team:NCTU Formosa/Notebook

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Wet Lab

July 1

Cloning

  • Received and resuspended of the IDT DNA fragments
  • Sample
    • GS linker+αS1-casein
    • Enterocin B
    • Enterocin 96
    • Bovicin HJ50
    • Durancin TW49M
    • Lacticin Z
    • Leucocyclicin Q

  • Received and resuspended of the IDT DNA fragments

July 2

None

July 3

Growth curve exp.

  • Observe the growth population of Bacillus subtilis in different temperature Condition: 37°C, 32°C, 27°C

July 4

Cloning gene of Curcumin bio-sensor

  • Doing the PCR and clean-up to amplify the DNA fragment. Digesting pET30a backbone and ligating it with αS1-casein+GS linker and T7 promoter.
  • Sample
    • pET30a backbone
    • DNA segment of αS1-casein+GS linker and T7 promoter.

July 5

Cloning gene of Curcumin bio-sensor Expression

  • Transforming the ligation product of the Curcumin bio-sensor into E. coli BL21 DE3 Cultivating the culture, preparing E. coli culture glycerol stocks, and doing the mini preparation.
  • Sample
    • fresh colony of E. coli BL21 DE3 carrying the backbone pET30a containing αS1-casein + GS linker and T7 promotor gene from an overnight plate

July 6

None

July 7

Expression of αS1-casein

  • Cultivating E. coli culture containing target protein αS1-casein from the E. coli glycerol stock and inducing it with IPTG to produce αS1-casein.
  • Purifying the protein by His-Tag.

July 8

Growth curve exp

  • Observe the growth population of Bacillus subtilis in different pH Condition: pH = 4, 5, 6, 7, 8

July 9

None

July 10

Chip Production

  • Preparing 18 chips for bio-sensor sensitivity pretest
    1. Dip the gold chips in 10mM Mua, RT for 4hrs.
    2. Wash the chips with 95% EtOH three times and dry.
    3. Add EDC+NHS mixture (100+100mM in DDW) on chips, RT for 1hrs.
    4. DDW rinse the chips and dry.
    5. Add αS1-casein on chips, RT for 1hrs.
    6. Wash with PBS three times and dry.
    6. Dip the chips in blocking solution, RT for 1.5hrs.
    7. Wash with PBS three times and dry.

July 11

Bio-sensor sensitivity pretest

  • Using DPV(Differential Pulse Voltammetry) to check whether our bio-sensor can detect curcumin.
    1. Add the diluted curcumin samples on our bio-sensor to react for 30min.
    2. Rinse with wash buffer and dry the chips.
    3. Wash the reference and counter electrodes with DDW, and dry them.
    4. Set up the three electrodes system within electrocheFunctional analysisal cell.
    5. Use the prototype of electrocheFunctional analysisal machine to measure the DPV method

July 12

Bio-sensor sensitivity assay –Determine Standard Curve and Create the Formula

  • Detecting the diluted standard curcumin samples by curcumin bio-sensor and made the standard curve and doing the polynomial curve fitting.

July 13

Bio-sensor sensitivity assay -Detecting the real Samples from Turmeric Root

  • Milling the turmeric root and dividing the powder into two groups. One of them was added with extraction buffer but not underwent the extraction protocol, and the other was added with extraction buffer but underwent the extraction process

July 14

None

July 15

Expression

  • Preparing competent cell E. coli ER2566

Growth curve exp

  • Observe the growth population of Bacillus subtilis in different salinity condition: 0.17M, 0.25M, 0.5M, 0.75M, 1.0M

July 16

Cloning

  • Amplify the insert gene (Pfu PCR)
  • Electrophoresis (To check PCR products)
  • Sample
    • Lacticin Z + pTXB1
    • Bovincin HJ50+ pTXB1

July 17

Cloning

  • Digestion of PCR products (Including insert and backbone pTXB1)
  • Ligation of digestion products (Ligase the DNA fragment into backbone pTXB1)
  • Sample
    • Lacticin Z + pTXB1
    • Bovincin HJ50+ pTXB1

July 18

Cloning

  • Transformation of ligation products (Transform into E. coli DH5α)
  • Taq PCR (PCR of ligation products to amplify insert gene)
  • Electrophoresis (To check PCR products)
  • Sample
    • Lacticin Z + pTXB1
    • Bovincin HJ50+ pTXB1

July 19

Cloning

  • Cultivation
  • Miniprep (Purify Plasmid)
  • Sample
    • Lacticin Z + pTXB1
    • Bovincin HJ50+ pTXB1

July 20

Cloning

  • Sequencing plasmid
  • Sample
    • Lacticin Z + pTXB1
    • Bovincin HJ50+ pTXB1

July 21

None

July 22

Cloning

  • Amplify the insert gene (Pfu PCR)
  • Electrophoresis (To check PCR products)
  • Digestion of PCR products (Including insert and backbone pTXB1)
  • Ligation of digestion products (Ligase the DNA fragment into backbone pTXB1)
  • Sample
    • Leucocyclicin Q + pTXB1

July 23

Cloning

  • Transformation of ligation products (Transform into E. coli DH5α)
  • Taq PCR (PCR of ligation products to amplify insert gene)
  • Electrophoresis (To check PCR products)
  • Sample
    • Leucocyclicin Q + pTXB1

July 24

Cloning

  • Cultivation
  • Miniprep (Purify Plasmid)
  • Sample
    • Leucocyclicin Q + pTXB1

July 25

Cloning

  • Sequencing plasmid
  • Sample
    • Leucocyclicin Q + pTXB1

July 26

None

July 27

None

July 28

None

July 29

Expression

  • Transformation (Transform correct plasmid into E. coli ER2566)
    • Leucocyclicin Q + pTXB1

July 30

None

July 31

None

August 1

None

August 2

None

August 3

None

August 4

None

August 5

Expression

  • Transformation (Transform correct plasmid into E. coli ER2566)
  • sample
    • Leucocyclicin Q + pTXB1

August 6

Expression

  • Cultivation (Cultivation of E. coli ER2566 colonies for IPTG induction)
  • IPTG Induction (To test the O.D. levels of E. coli ER2566 to induce)Condition: O.D. = 0.5, 1.3
  • sample
    • Leucocyclicin Q + pTXB1

August 7

Expression

  • SDS-PAGE (To check the protein production after induction)
  • sample
    • Leucocyclicin Q + pTXB1

August 8

None

August 9

None

August 10

None

August 11

Cloning

  • Amplify the insert gene (Pfu PCR)
  • Electrophoresis (To check PCR products)
  • sample
    • Enterocin B
    • Enterocin 96
    • Durancin TW49M

August 12

Cloning

  • Digestion of PCR products (Including insert and backbone pTXB1)
  • Ligation of digestion products (Ligase the DNA fragment into backbone pTXB1)
  • Transformation of ligation products (Transform into E. coli DH5α)
  • sample
    • Enterocin B + pTXB1
    • Enterocin 96 + pTXB1
    • Durancin TW-49M + pTXB1

Growth curve

  • Observe the growth of Bacillus subtilis in different temperature, pH, salinityCondition:
  • 1.Temp: 37°C , pH:7 , salinity: 0.17M
  • 2.Temp: 30°C , pH:9 , salinity: 0.5M
  • 3.Temp: 25°C , pH:5 , salinity: 0.25M

August 13

Cloning

  • Taq PCR (PCR of ligation products to amplify insert gene)
  • Electrophoresis (To check PCR products)
  • sample
    • Enterocin B + pTXB1
    • Enterocin 96 + pTXB1
    • Durancin TW-49M + pTXB1

August 14

Cloning

  • Cultivation
  • Miniprep (Purify Plasmid)
  • Sample
    • Enterocin B + pTXB1
    • Enterocin 96 + pTXB1
    • Durancin TW-49M + pTXB1

Expression

  • Transformation (Transform correct plasmid into E. coli ER2566)to test different cultivation temperature

August 15

Cloning

  • Sequencing plasmid
  • Sample
    • Enterocin B + pTXB1
    • Enterocin 96 + pTXB1
    • Durancin TW-49M + pTXB1

Expression

  • Cultivation (Cultivation of E. coli ER2566 colonies for IPTG induction)
  • IPTG Induction (To test the temperature of cultivation after induction)Condition: temp = 37, 30, 13.5°C

August 16

Expression

  • SDS-PAGE (To check the protein production after induction)Check different temperature
  • IPTG Induction (To test the concentration of IPTG)Condition: concentration = 200, 400, 600, 800, 1000μM

August 17

Expression

  • SDS-PAGE (To check the protein production after induction) -> Check different IPTG concentration

August 18

Transformation

  • Transforming backbone pTXB1 into E. coli ER2566 to produce protein as our negative control in verifying the function of our target peptide.
  • Transforming backbone pTXB1 containing bacteriocin gene into E. coli ER2566 to produce our target protein as a bio-stimulator.
  • Sample
    • backbone pTXB1 mini
    • backbone pTXB1 containing Enterocin B gene mini
    • backbone pTXB1 containing Enterocin 96 gene mini
    • backbone pTXB1 containing Leucocyclicin Q gene mini

    Cultivation

    • Cultivate the E. coli ER2566 at 37°C cultivating Bacillus subtilis ER2566 at 37°C carrying the backbone pTXB1 to produce protein as our negative control in verifying the function of our target peptide.
    • Cultivate the E. coli ER2566 at 37°C cultivating E. coli ER2566 at 37°C carrying the backbone pTXB1 containing bacteriocin gene to produce our target protein as a bio-stimulator.
    • Sample
      • Fresh colony of E. coli ER2566 carrying the backbone pTXB1 from an overnight plate
      • Fresh colony of E. coli ER2566 carrying the backbone pTXB1 containing Enterocin B gene from an overnight plate
      • Fresh colony of E. coli ER2566 carrying the backbone pTXB1 containing Enterocin 96 gene from an overnight plate
      • Fresh colony of E. coli ER2566 carrying the backbone pTXB1 containing Leucocyclicin Q gene from an overnight plate

      IPTG Induction

      • Inducting E. coli ER2566 carrying the backbone pTXB1 after cultivating at 37°C to produce protein as our negative control in verifying the function of our target peptide
      • Inducting E. coli ER2566 carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C to produce our target protein as a bio-stimulator.
      • Sample
        • Culture of E. coli ER2566 carrying the backbone pTXB1
        • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Enterocin B gene
        • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Enterocin 96 gene
        • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Leucocyclicin Q gene

August 19

Cloning

  • Digestion of PCR products (Including insert and backbone pET30a)
  • Ligation of digestion products (Ligase the DNA fragment into backbone pET30a)
  • Sample(8/17)
    • Enterocin B + pET30a
    • Enterocin 96 + pET30a
    • Bovicin HJ50 + pET30a
    • Durancin TW-49M + pET30a
    • Lacticin Z + pET30a
    • Leucocyclicin Q + pET30a

Sonication

  • Breaking E. coli ER2566 carrying the backbone pTXB1 and pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
  • Sample
    • Culture of E. coli ER2566 carrying the backbone pTXB1 induced by IPTG
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Enterocin B gene induced by IPTG
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Enterocin 96 gene induced by IPTG
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Leucocyclicin Q gene induced by IPTG

Protein quantification & SDS-PAGE

  • Quantificating the protein expressing the backbone pTXB1 and pTXB1 containing bacteriocin gene by Bradford method and running SDS-PAGE to check the protein expression.
  • Sample
    • Protein expressing the backbone pTXB1 induced by IPTG
    • Protein expressing the backbone pTXB1 containing Enterocin B gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Enterocin 96 gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Leucocyclicin Q gene induced by IPTG

August 20

Cloning

  • Transformation of ligation products (Transform into E. coli DH5α)
  • Taq PCR (PCR of ligation products to amplify insert gene)
  • Electrophoresis (To check PCR products)
  • Sample
    • Enterocin B + pET30a
    • Enterocin 96 + pET30a
    • Bovicin HJ50 + pET30a
    • Durancin TW-49M + pET30a
    • Lacticin Z + pET30a
    • Leucocyclicin Q + pET30a

August 21

Cloning

  • Cultivation
  • Miniprep (Purify Plasmid)
  • Sample
    • Enterocin B + pET30a
    • Enterocin 96 + pET30a
    • Bovicin HJ50 + pET30a
    • Durancin TW-49M + pET30a
    • Lacticin Z + pET30a
    • Leucocyclicin Q + pET30a

August 22

Cloning

  • Sequencing plasmid
  • Sample
    • Enterocin B + pET30a
    • Enterocin 96 + pET30a
    • Bovicin HJ50 + pET30a
    • Durancin TW-49M + pET30a
    • Lacticin Z + pET30a
    • Leucocyclicin Q + pET30a

August 23

None

August 24

Transformation

  • Transforming backbone pTXB1 containing bacteriocin gene into E. coli ER2566 to produce our target protein as a bio-stimulator.
  • Sample
    • backbone pTXB1 containing Bovicin HJ50 gene mini
    • backbone pTXB1 containing Durancin TW-49M gene mini

Cultivation

  • Cultivate the E. coli ER2566 at 37°C cultivating E. coli ER2566 at 37°C carrying the backbone pTXB1 containing bacteriocin gene to produce our target protein as a bio-stimulator.
  • Sample
    • Fresh colony of E. coli ER2566 carrying the backbone pTXB1 containing Bovicin HJ50 gene from an overnight plate
    • Fresh colony of E. coli ER2566 carrying the backbone pTXB1 containing Durancin TW-49M gene from an overnight plate

IPTG Induction

  • Inducting E. coli ER2566 carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C to produce our target protein as a bio-stimulator.
  • Sample
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Bovicin HJ50 gene
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Durancin TW-49M gene

August 25

Sonication

  • Breaking E. coli ER2566 carrying the backbone pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
  • Sample
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Bovicin HJ50 gene induced by IPTG
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Durancin TW-49M gene induced by IPTG

Protein quantification & SDS-PAGE

  • Quantificating the protein expressing the backbone pTXB1 containing bacteriocin gene by Bradford method and running SDS-PAGE to check the protein expression.
  • Sample
    • Protein expressing the backbone pTXB1 containing Bovicin HJ50 gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Durancin TW-49M gene induced by IPTG

August 26

None

August 27

None

August 28

None

August 29

None

August 30

None

August 31

None

September 1

None

September 2

None

September 3

None

September 4

None

September 5

None

September 6

Transformation

  • Transforming backbone pTXB1 containing Lacticin Z gene into E. coli ER2566 to produce our target protein as a bio-stimulator.
  • Sample
    • backbone pTXB1 containing Lacticin Z gene mini

Cultivation

  • Cultivate the E. coli ER2566 at 37°C cultivating Bacillus subtilis ER2566 at 37°C carrying the backbone pTXB1 containing Lacticin Z gene to produce our target protein as a bio-stimulator.
  • Sample
    • Fresh colony of E. coli ER2566 carrying the backbone pTXB1 containing Lacticin Z gene from an overnight plate

IPTG Induction

  • Inducting E. coli ER2566 carrying the backbone pTXB1 containing Lacticin Z gene after cultivating at 37°C to produce our target protein as a bio-stimulator.
  • Sample
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Lacticin Z gene

September 7

Sonication

  • Breaking E. coli ER2566 carrying the backbone pTXB1 containing Lacticin Z gene after cultivating to harvest the protein by sonication.
  • Sample
    • Culture of E. coli ER2566 carrying the backbone pTXB1 containing Lacticin Z gene induced by IPTG

Protein quantification & SDS-PAGE

  • Quantificating the protein expressing the backbone pTXB1 containing Lacticin Z gene by Bradford method and running SDS-PAGE to check the protein expression.
  • Sample
    • Protein expressing the backbone pTXB1 containing Lacticin Z gene induced by IPTG

September 8

None

September 9

None

September 10

None

September 11

None

September 12

None

September 13

None

September 14

None

September 15

None

September 16

None

September 17

None

September 18

None

September 19

None

September 20

None

September 21

Functional analysis test

  • Test the function of Lacticin Z (produced by E. coli ER2566) by elisa reader(triplicate)
  • Sample
    • Control:
      1. Bacillus subtilis + Ampicillin
      2. Bacillus subtilis + Production of the pTXB1 backbone induced by IPTG
      3. Bacillus subtilis + LB broth
      4. LB broth for background
    • Test:
      1. Bacillus subtilis + Production of the pTXB1 backbone containing Lacticin Z gene induced by IPTG

September 22

None

September 23

None

September 24

None

September 25

None

September 26

None

September 27

None

September 28

None

September 29

None

September 30

None

October 1

None

October 2

None

October 3

None

October 4

None

October 5

Transformation

  • Transforming backbone pTXB1 into E. coli Rosetta-Gami to produce protein as our negative control in verifying the function of our target peptide.
  • Transforming backbone pTXB1 containing bacteriocin gene into E. coli Rosetta-Gami to produce our target protein as a bio-stimulator.
  • Sample
    • backbone pTXB1 mini
    • backbone pTXB1 containing Enterocin B gene mini
    • backbone pTXB1 containing Enterocin 96 gene mini
    • backbone pTXB1 containing Bovicin HJ50 gene mini
    • backbone pTXB1 containing Durancin TW-49M gene mini
    • backbone pTXB1 containing Lacticin Z gene mini
    • backbone pTXB1 containing Leucocyclicin Q gene mini

October 6

Cultivation

  • Cultivate the E. coli ER2566 at 37°C cultivating E. coli Rosetta-Gami at 37°C carrying the backbone pTXB1 to produce protein as our negative control in verifying the function of our target peptide.
  • Cultivate the E. coli ER2566 at 37°C cultivating E. coli Rosetta-Gami at 37°C carrying the backbone pTXB1 containing bacteriocin gene produce our target protein as a bio-stimulator.
  • Sample
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 from an overnight plate
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Enterocin B gene from an overnight plate
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Enterocin 96 gene from an overnight plate
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Leucocyclicin Q gene from an overnight plate

IPTG Induction

  • Cultivate the E. coli ER2566 at 37°C cultivating E. coli Rosetta-Gami at 37°C carrying the backbone pTXB1 to produce protein as our negative control in verifying the function of our target peptide.
  • Inducting E. coli Rosetta-Gami carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C produce our target protein as a bio-stimulator.
  • Sample
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 from an overnight plate
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Enterocin B gene
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Enterocin 96 gene
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Leucocyclicin Q gene

October 7

Cultivation

  • Cultivate the E. coli ER2566 at 37°C cultivating E. coli Rosetta-Gami at 37°C carrying the backbone pTXB1 containing bacteriocin gene produce our target protein as a bio-stimulator.
  • Sample
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Bovicin HJ50 gene from an overnight plate
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Durancin TW-49M gene from an overnight plate
    • Fresh colony of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Lacticin Z gene from an overnight plate

IPTG Induction

  • Inducting E. coli Rosetta-Gami carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C produce our target protein as a bio-stimulator.
  • Sample
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Bovicin HJ50 gene
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Durancin TW-49M gene
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Lacticin Z gene

Sonication

  • Breaking E. coli Rosetta-Gami carrying the backbone pTXB1 and pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
  • Sample
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 induced by IPTG
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Enterocin B gene induced by IPTG
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Enterocin 96 gene induced by IPTG
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Leucocyclicin Q gene induced by IPTG

Protein quantification & SDS-PAGE

  • Quantificating the protein expressing the backbone pTXB1 and pTXB1 containing bacteriocin gene by Bradford method and running SDS-PAGE to check the protein expression.
  • Sample
    • Protein expressing the backbone pTXB1 induced by IPTG
    • Protein expressing the backbone pTXB1 containing Enterocin B gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Enterocin 96 gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Leucocyclicin Q gene induced by IPTG

October 8

Sonication

  • Breaking E. coli Rosetta-Gami carrying the backbone pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
  • Sample
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Bovicin HJ50 gene induced by IPTG
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Durancin TW-49M gene induced by IPTG
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Lacticin Z gene induced by IPTG
    • Culture of E. coli Rosetta-Gami carrying the backbone pTXB1 containing Leucocyclicin Q gene induced by IPTG

Protein quantification & SDS-PAGE

  • Quantificating the protein expressing the backbone pTXB1 containing bacteriocin gene by Bradford method and running SDS-PAGE to check the protein expression.
  • Sample
    • Protein expressing the backbone pTXB1 containing Bovicin HJ50 gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Durancin TW-49M gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Lacticin Z gene induced by IPTG
    • Protein expressing the backbone pTXB1 containing Leucocyclicin Q gene induced by IPTG

October 9

Functional analysis test

  • Test the function of bacteriocins (produced by E. coli Rosetta-gami) by elisa reader(triplicate)
  • Sample
    • Control:
      1. Bacillus subtilis + Ampicillin
      2. Bacillus subtilis + Production of the pTXB1 backbone induced by IPTG
      3. Bacillus subtilis + LB broth
      4. LB broth for background
    • Test:
      1. Bacillus subtilis + Production of the pTXB1 backbone containing Enterocin B gene induced by IPTG
      2. Bacillus subtilis + Production of the pTXB1 backbone containing Enterocin 96 gene induced by IPTG
      3. Bacillus subtilis + Production of the pTXB1 backbone containing Lacticin Z gene induced by IPTG
      4. Bacillus subtilis + Production of the pTXB1 backbone containing Durancin TW-49M gene induced by IPTG

October 10

Functional analysis test

  • Test the function of bacteriocins (produced by E. coli Rosetta-gami) by elisa reader(triplicate)
  • Sample
    • Control:
      1. Bacillus subtilis + Ampicillin
      2. Bacillus subtilis + Production of the pTXB1 backbone induced by IPTG
      3. Bacillus subtilis + LB broth
      4. LB broth for background
    • Test:
      1. Bacillus subtilis + Production of the pTXB1 backbone containing Bovicin HJ50 gene induced by IPTG
      2. Bacillus subtilis + Production of the pTXB1 backbone containing Leucocyclicin Q gene induced by IPTG

October 11

None

October 12

Functional analysis test

  • Test the function of bacteriocins (produced by E. coli Rosetta-gami) by elisa reader(Dose response assessment)
  • Sample
    • Control:
      1. Bacillus subtilis + Ampicillin
      2. Bacillus subtilis + Production of the pTXB1 backbone induced by IPTG
      3. Bacillus subtilis + LB broth
      4. LB broth for background
    • Test:
      1. Bacillus subtilis + Production of the pTXB1 backbone containing Enterocin B gene induced by IPTG
      2. Bacillus subtilis + Production of the pTXB1 backbone containing Enterocin 96 gene induced by IPTG
      3. Bacillus subtilis + Production of the pTXB1 backbone containing Lacticin Z gene induced by IPTG
      4. Bacillus subtilis + Production of the pTXB1 backbone containing Durancin TW-49M gene induced by IPTG

October 13

None

October 14

None

October 15

Functional analysis test

  • Test the function of bacteriocins (produced by E. coli Rosetta-gami) by elisa reader(Dose response assessment)
  • Sample
    • Control:
      1. Bacillus subtilis + Ampicillin
      2. Bacillus subtilis + Production of the pTXB1 backbone induced by IPTG
      3. Bacillus subtilis + LB broth
      4. LB broth for background
    • Test:
      1. Bacillus subtilis + Production of the pTXB1 backbone containing Bovicin HJ50 gene induced by IPTG
      2. Bacillus subtilis + Production of the pTXB1 backbone containing Leucocyclicin Q gene induced by IPTG

Functional analysis test(inhibition zone)

  • Test the function of Enterocin B (produced by E. coli Rosetta-gami) by LB Agar + Ampicillin plate
  • Sample
    • The Bacillus subtilis on the plate
    • Control:
      1. Ampicillin
      2. Production of the pTXB1 backbone induced by IPTG
    • Test:
      1. Production of the pTXB1 backbone containing Enterocin B gene induced by IPTG

October 16

Functional analysis test(inhibition zone)

  • Test the function of bacteriocins (produced by E. coli Rosetta-gami) by LB Agar + Ampicillin plate
  • Sample
    • The Bacillus subtilis on the plate
    • Control:
      1. Ampicillin
      2. Production of the pTXB1 backbone induced by IPTG
    • Test:
      1. Production of the pTXB1 backbone containing Bovicin HJ50 gene induced by IPTG
      2. Production of the pTXB1 backbone containing Lacticin Z gene induced by IPTG

October 17

Functional analysis test

  • Test the function of bacteriocins (produced by E. coli Rosetta-gami) by elisa reader(Dose response assessment)
  • Sample
    • Control:
      1. Bacillus subtilis + Ampicillin
      2. Bacillus subtilis + Production of the pTXB1 backbone induced by IPTG
      3. Bacillus subtilis + LB broth
    • Test:
      1. Bacillus subtilis + Production of the pTXB1 backbone containing Durancin TW-49M gene induced by IPTG

October 18

None

October 19

None