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 biosensor

• 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 biosensor Expression

• Transforming the ligation product of the Curcumin biosensor 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 biosensor 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

Biosensor sensitivity pretest

• Using DPV(Differential Pulse Voltammetry) to check whether our biosensor can detect curcumin.
  1. Add the diluted curcumin samples on our biosensor 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 electrochemical cell.
  5. Use the prototype of electrochemical machine to measure the DPV method

July 12

Biosensor sensitivity assay –Determine Standard Curve and Create the Formula

• Detecting the diluted standard curcumin samples by curcumin biosensor and made the standard curve and doing the polynomial curve fitting.

July 13

Biosensor 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 TW49M + 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 TW49M + pTXB1

August 14

Cloning

• Cultivation
• Miniprep (Purify Plasmid)
• Sample
• Enterocin B + pTXB1
• Enterocin 96 + pTXB1
• Durancin TW49M + 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 TW49M + 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 Bacillus subtilis ER2566 to produce protein as our negative control in verifying the function of our target peptide.
• Transforming backbone pTXB1 containing bacteriocin gene into Bacillus subtilis ER2566 to produce our target protein as a biostimulator.
• 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 Bacillus subtilis ER2566 at 37°C carrying the backbone pTXB1 containing bacteriocin gene to produce our target protein as a biostimulator.
• Sample
• Fresh colony of Bacillus subtilis ER2566 carrying the backbone pTXB1 from an overnight plate
• Fresh colony of Bacillus subtilis ER2566 carrying the backbone pTXB1 containing Enterocin B gene from an overnight plate
• Fresh colony of Bacillus subtilis ER2566 carrying the backbone pTXB1 containing Enterocin 96 gene from an overnight plate
• Fresh colony of Bacillus subtilis ER2566 carrying the backbone pTXB1 containing Leucocyclicin Q gene from an overnight plate

IPTG Induction

• Inducting Bacillus subtilis 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 Bacillus subtilis ER2566 carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C to produce our target protein as a biostimulator.
• Sample
• Culture of Bacillus subtilis carrying the backbone pTXB1
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Enterocin B gene
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Enterocin 96 gene
• Culture of Bacillus subtilis 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 TW49M + pET30a
• Lacticin Z + pET30a
• Leucocyclicin Q + pET30a

Sonication

• Breaking Bacillus subtilis ER2566 carrying the backbone pTXB1 and pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
• Sample
• Culture of Bacillus subtilis carrying the backbone pTXB1 induced by IPTG
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Enterocin B gene induced by IPTG
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Enterocin 96 gene induced by IPTG
• Culture of Bacillus subtilis 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 TW49M + 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 TW49M + pET30a
• Lacticin Z + pET30a
• Leucocyclicin Q + pET30a

August 22

Cloning

• Sequencing plasmid
• Sample
• Enterocin B + pET30a
• Enterocin 96 + pET30a
• Bovicin HJ50 + pET30a
• Durancin TW49M + pET30a
• Lacticin Z + pET30a
• Leucocyclicin Q + pET30a

August 23

None

August 24

Transformation

• Transforming backbone pTXB1 containing bacteriocin gene into Bacillus subtilis ER2566 to produce our target protein as a biostimulator.
• Sample
• backbone pTXB1 containing Bovicin HJ50 gene mini
• backbone pTXB1 containing Durancin gene mini

Cultivation

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

IPTG Induction

• Inducting Bacillus subtilis ER2566 carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C to produce our target protein as a biostimulator.
• Sample
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Bovicin HJ50 gene
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Durancin gene

August 25

Sonication

• Breaking Bacillus subtilis ER2566 carrying the backbone pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
• Sample
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Bovicin HJ50 gene induced by IPTG
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Durancin 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 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 Bacillus subtilis ER2566 to produce our target protein as a biostimulator.
• 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 biostimulator.
• Sample
• Fresh colony of Bacillus subtilis ER2566 carrying the backbone pTXB1 containing Lacticin Z gene from an overnight plate

IPTG Induction

• Inducting Bacillus subtilis ER2566 carrying the backbone pTXB1 containing Lacticin Z gene after cultivating at 37°C to produce our target protein as a biostimulator.
• Sample
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Lacticin Z gene

September 7

Sonication

• Breaking Bacillus subtilis ER2566 carrying the backbone pTXB1 containing Lacticin Z gene after cultivating to harvest the protein by sonication.
• Sample
• Culture of Bacillus subtilis 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

None

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 Bacillus subtilis Rosetta-Gami to produce protein as our negative control in verifying the function of our target peptide.
• Transforming backbone pTXB1 containing bacteriocin gene into Bacillus subtilis Rosetta-Gami to produce our target protein as a biostimulator.
• 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 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°Ccultivating Bacillus subtilis 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°Ccultivating Bacillus subtilis Rosetta-Gami at 37°C carrying the backbone pTXB1 containing bacteriocin gene produce our target protein as a biostimulator.
• Sample
• Fresh colony of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 from an overnight plate
• Fresh colony of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Enterocin B gene from an overnight plate
• Fresh colony of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Enterocin 96 gene from an overnight plate
• Fresh colony of Bacillus subtilis 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 Bacillus subtilis 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 Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C produce our target protein as a biostimulator.
• Sample
• Fresh colony of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 from an overnight plate
• Culture of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Enterocin B gene
• Culture of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Enterocin 96 gene
• Culture of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Leucocyclicin Q gene

October 7

Cultivation

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

IPTG Induction

• Inducting Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing bacteriocin gene after cultivating at 37°C produce our target protein as a biostimulator.
• Sample
• Culture of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Bovicin HJ50 gene
• Culture of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Durancin gene
• Culture of Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing Lacticin Z gene

Sonication

• Breaking Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 and pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
• Sample
• Culture of Bacillus subtilis carrying the backbone pTXB1 induced by IPTG
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Enterocin B gene induced by IPTG
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Enterocin 96 gene induced by IPTG
• Culture of Bacillus subtilis 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 Bacillus subtilis Rosetta-Gami carrying the backbone pTXB1 containing bacteriocin gene after cultivating to harvest the protein by sonication.
• Sample
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Bovicin HJ50 gene induced by IPTG
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Durancin gene induced by IPTG
• Culture of Bacillus subtilis carrying the backbone pTXB1 containing Lacticin Z gene induced by IPTG
• Culture of Bacillus subtilis 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 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

None

October 10

None

October 11

None

October 12

None

October 13

None

October 14

None

October 15

None

October 16

None

October 17

None

October 18

None

October 19

None