July 2

None

July 3

Growth curve exp

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

July 4

None

July 5

None

July 6

None

July 7

None

July 8

Growth curve exp

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

July 9

None

July 10

None

July 11

None

July 12

None

July 13

None

July 14

None

July 15

Expression

• repared competent cell E. coli ER2566

Growth curve exp

• Observe the growth population of Bacillis subtilis in different salinity Condition: 0.17M, 0.25M, 0.5M, 0.75M, 1.0M
• Sample

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

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

July 21

Cultivation

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

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

August 6

Expression

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

August 7

Expression

• Purepose: 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

• Purepose: Amplify the insert gene (Pfu PCR)
• Purepose: Electrophoresis (To check PCR products)
• sample
• Enterocin A
• Enterocin B
• Enterocin 96
• Duracin TW49M
• Subtilosin

August 12

Cloning

• Purepose: Digestion of PCR products (Including insert and backbone pTXB1)
• Purepose: Ligation of digestion products (Ligase the DNA fragment into backbone pTXB1)
• Purepose: Transformation of ligation products (Transform into E. coli DH5α)
• sample
• Enterocin A + pTXB1
• Enterocin B + pTXB1
• Enterocin 96 + pTXB1
• Duracin TW49M + pTXB1
• Subtilosin + pTXB1

Growth curve

• Purepose: 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

• Purepose: Taq PCR (PCR of ligation products to amplify insert gene)
• Purepose: Electrophoresis (To check PCR products)
• sample
• Enterocin A + pTXB1
• Enterocin B + pTXB1
• Enterocin 96 + pTXB1
• Duracin TW49M + pTXB1
• Subtilosin + pTXB1

August 14

Cloning

• Cultivation
• Miniprep (Purify Plasmid)
• Sample
• Enterocin A + pTXB1
• Enterocin B + pTXB1
• Enterocin 96 + pTXB1
• Duracin TW49M + pTXB1
• Subtilosin + pTXB1

Expression

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

August 15

Cloning

• Purepose: Sequencing plasmid
• Sample
• Enterocin A + pTXB1
• Enterocin B + pTXB1
• Enterocin 96 + pTXB1
• Duracin TW49M + pTXB1
• Subtilosin + pTXB1

Expression

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

August 16

Expression

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

August 17

Expression

• Purepose: SDS PAGE (To check the protein production after induction)Check different IPTG concentration
• Sample

August 18

SDS-PAGE

August 19

Cloning

• Purepose: Digestion of PCR products (Including insert and backbone pET30a)
• Purepose: Ligation of digestion products (Ligase the DNA fragment into backbone pET30a)
• Sample(8/17)
• Enterocin A + pET30a
• Enterocin B + pET30a
• Enterocin 96 + pET30a
• Bovicin HJ50 + pET30a
• Duracin TW49M + pET30a
• Lacticin Z + pET30a
• Leucocyclicin Q + pET30a
• Subtilosin + pET30a

August 20

Cloning

• Purepose: Transformation of ligation products (Transform into E. coli DH5α)
• Purepose: Taq PCR (PCR of ligation products to amplify insert gene)
• Purepose: Electrophoresis (To check PCR products)
• Sample
• Enterocin A + pET30a
• Enterocin B + pET30a
• Enterocin 96 + pET30a
• Bovicin HJ50 + pET30a
• Duracin TW49M + pET30a
• Lacticin Z + pET30a
• Leucocyclicin Q + pET30a
• Subtilosin + pET30a

August 21

Cloning

• Purepose: Cultivation
• Purepose: Miniprep (Purify Plasmid)
• Sample
• Enterocin A + pET30a
• Enterocin B + pET30a
• Enterocin 96 + pET30a
• Bovicin HJ50 + pET30a
• Duracin TW49M + pET30a
• Lacticin Z + pET30a
• Leucocyclicin Q + pET30a
• Subtilosin + pET30a

August 22

Cloning

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

August 23

Protein purification(8/22-8/23)

• Sample
• Hv1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

Cultivation and IPTG induction

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r 8/20
• Sf1a-lectin-his A^r 8/20
• Add 1000μM IPTG and test OD₆₀₀

Sonication

• Sample
• Hv1a-his A^r 8/23
• Sf1a-lectin-his A^r 8/23

August 24

Protein purification(8/23-8/24)

• Sample
• Hv1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

Cultivation and IPTG induction

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Sf1a-his A^r 8/20
• Sf1a-lectin-his A^r 8/20
• Refresh to approximately OD₆₀₀ 0.7
• Add 1000μM IPTG and test OD₆₀₀
• Sf1a-his: 0.966
• Sf1a-lectin-his: 1.363
• Certification, and add 2mL binding buffer and 200μM PMSF for sonication. Repeat three times.

Sonication

• Sample
• Sf1a-his A^r 8/24
• Sf1a-lectin-his A^r 8/24

SDS-PAGE

• Check the purification protein.
• Sample
• Hv1a-lectin-his(8/22-8/23)

August 25

Protein purification(8/24-8/25)

• Sample
• Hv1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample
• Hv1a-lectin-his(8/23-8/24)

August 26

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r 8/15
• Hv1a-lectin-his A^r 8/15
• Sf1a-lectin-his A^r 8/15
• OAIP-lectin-his A^r 8/15

Sonication

• Sample
• Sf1a-his A^r 8/24
• Sf1a-lectin-his A^r 8/24

Protein purification

• Sample
• Hv1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Sf1a-his A^r 8/17
• OAIP-his A^r 8/17

IPTG induction

• Induce the mass protein expression for SDS-PAGE.
• The protein would form inclusion body. Therefore, it was devided into two parts, the urea and control.
• Sample
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26
• Sf1a-lectin-his A^r 8/26
• OAIP-lectin-his A^r 8/26
• Add 500μM IPTG
• Take the following samples to go on the experience first. The others storage in 4℃ refrigerator.
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26

SDS-PAGE

• Check the protein in pellet or in solution.
• Sample
• Hv1a-his A^r 8/26(with urea)
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26(with urea)
• Hv1a-lectin-his A^r 8/26
• Sonication and the supernatant is loading sample1
• 10 seconds on, 40 seconds off and repeat six times.
• Add binding buffer (with/without urea) again and the solution is loading sample 2
• Sonication again and the supernatant is loading sample 3
• 10 seconds on, 40 seconds off and repeat six times.
• Add binding buffer (with/without urea) again and the solution is loading sample4
• Loading sample
• Hv1a-his (with urea) 1~4
• Hv1a-his 1~4
• Hv1a-lectin-his (with urea) 1~4
• Hv1a-lectin-his 1~4

IPTG induction

• Induce the mass protein expression for SDS-PAGE.
• The protein would form inclusion body. Therefore, it was devided into two parts, the urea and control.
• Sample
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26
• Sf1a-lectin-his A^r 8/26
• OAIP-lectin-his A^r 8/26
• Add 500μM IPTG and test OD₆₀₀ to approximately 1.050

SDS-PAGE

• Check the protein in pellet or in solution.
• Sample
• Hv1a-his A^r 8/26(with urea)
• Hv1a-his A^r 8/26
• Hv1a-lectin-his A^r 8/26(with urea)
• Hv1a-lectin-his A^r 8/26
• Sf1a-lectin-his A^r 8/26(with urea)
• Sf1a-lectin-his A^r 8/26
• OAIPlectin-his A^r 8/26(with urea)
• OAIP-lectin-his A^r 8/26
• Certificate the sample, add binding buffer (with/without urea), PMSF
• Sonication and the supernatant is loading sample1
• 10 seconds on, 40 seconds off and repeat six times.
• Add binding buffer (with/without urea) again and the solution is loading sample 2
• Sonication again and the supernatant is loading sample 3
• 10 seconds on, 40 seconds off and repeat six times.
• Add binding buffer (with/without urea) again and the solution is loading sample4
• Loading sample
• Hv1a-his (with urea) 1~4
• Hv1a-his 1~4
• Hv1a-lectin-his (with urea) 1~4
• Hv1a-lectin-his 1~4
• Sf1a-lectin-his (with urea) 1~4
• Sf1a-lectin-his 1~4
• OAIPlectin-his (with urea) 1~4
• OAIP-lectin-his 1~4

August 27

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r

August 28

Protein purification

• The buffer contains the urea
• Sample(8/27)
• Hv1a-his
• Hv1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample(8/28)
• Hv1a-his
• Hv1a-lectin-his

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Sf1a-his A^r 8/17

August 29

Protein purification

• The buffer contains the urea
• Sample(8/28)
• Sf1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample(8/29)
• Sf1a-lectin-his

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Sf1a-his A^r 8/17

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r 8/15

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-lectin-his A^r 8/15
• Sf1a-lectin-his A^r 8/15
• OAIP-lectin-his A^r 8/15

August 30

IPTG induction

• Induce the mass protein expression for SDS-PAGE.
• The protein would form inclusion body. Therefore, it was devided into two parts, the urea and control.
• Sample
• Hv1a-lectin-his A^r 8/29
• Sf1a-lectin-his A^r 8/29
• OAIP-lectin-his A^r 8/29
• Refresh to approximately OD₆₀₀ 0.6
• Add 500μM IPTG and cultivate for 4hr

Protein purification

• The buffer contains the urea
• Sample(8/30)
• Sf1a-lectin-his
• Hv1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample(8/29)
• Sf1a-lectin-his
• Hv1a-lectin-his

PCR

• amplify the insert gene
• Sample(8/30)
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

Electrophoresis

• Electrophoresis of PCR product
• Sample(8/30 PCR product)
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

Digestion

• Digest min for transformation
• Sample
• Hv1a-his digestion EP
• Hv1a-lectin-his digestion ES
• Sf1a-his digestion EP
• Sf1a-lectin-his digestion ES
• OAIP-his digestion EP
• OAIP-lectin-his digestion ES

Transformation

• Transformation PSB1C3 to DH5α
• Sample
• Trans PSB1C3 8/30
• Trans PSB1C3 8/30

Urea test

• Check whether protein in inclusion body or not.
• Sample
• Hv1a-his
• Hv1a-his + Urea
• Sf1a-his
• Sf1a-his + Urea
• OAIP-his
• OAIP-his + Urea

August 31

Ligation

• Ligase insert gene into the pSB1C3
• Sample
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

Transformation

• Transformation of ligated product to DH5α
• Sample(8/31 Ligation product)
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

Dialysis and refolding

• Dialysis and refolding for insoluble protein
• Sample(8/8)
• Hv1a-lectin-his (8/30)
• Sf1a-lectin-his(8/30)
• OAIP-lectin-his(8/30)
• Hv1a-his(8/28)

SDS-PAGE

• Check the purification protein.
• Sample(8/30)
• Sf1a-lectin-his
• Hv1a-lectin-his

September 1

Protein purification

• The buffer contains the urea
• Sample(8/30)
• OAIP-lectin-his
• Hv1a-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample(8/30)
• OAIP-lectin-his
• Hv1a-his

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• OAIP-his A^r
• Sf1a-his A^r

IPTG Induction

• Prepare for the purify the protein from sample BL21-Rosetta-gami colonies.
• Sample
• Hv1a-his A^r
• OAIP-his A^r
• Sf1a-his A^r
• Add IPTG 500μM and test OD₆₀₀ after 4hr.

Sonication

• Sample
• Hv1a-his A^r
• OAIP-his A^r
• Sf1a-his A^r

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Sonication

• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

September 2

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

Protein quantification Braford method

• Sample
• Hv1a-his
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Dialysis and refolding

• Dialysis and refolding for insoluble protein
• Sample
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

September 3

Protein purification

• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

Protein quantification Braford method

• The buffer contains the urea
• Sample
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Concentrate protein

• Increase the concentration of protein
• Sample
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

September 4

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

September 5

IPTG Induction

• Prepare for the purify the protein from sample BL21-Rosetta-gami colonies.
• Sample
• Hv1a-his
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• Add IPTG 500μM and test OD₆₀₀ after 4hr.

Sonication

• Sample
• Hv1a-his
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Protein purification

• The buffer contains the urea
• Sample
• Hv1a-his
• Hv1a-lectin-his
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

UV test

• Test degradation rate
• Sample
• Hv1a-his
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Concentrate protein

• Increase the concentration of protein
• Sample
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

Protein quantification Braford method

• Sample
• Hv1a-lectin-his A^r
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r

September 6

Protein purification

• The buffer contains the urea
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 7

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-lectin-his A^r
• OAIP-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample
• OAIP-lectin-his A^r
• Sf1a-lectin-his A^r

UV test

• Test degradation rate
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Cultivation

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Sonication

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 8

Protein purification

• The buffer contains the urea
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r

Cultivation and IPTG induction

• Cultivation of sample BL21-Rosetta-gami colonies for protein purification.
• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r
• Refresh to approximately OD₆₀₀ 0.7
• Add 1000μM IPTG and test OD₆₀₀
• Hv1a-his:0.772
• Hv1a-lectin-his:0.631
• Sf1a-his:0.567
• Sf1a-lectin-his:0.78
• OAIP-his:0.851
• OAIP-lectin-his:0.819
• Certification, and add 2mL binding buffer and 200μM PMSF for sonication. Repeat three times.

Sonication

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Sonication

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 9

Protein purification

• The buffer contains the urea
• Sample
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r
• Collect：
  pass through solution, wash through solution(W1~3), elution solution(E1~10), filtered NaCl solution, and filtered ddH₂O solution.

SDS-PAGE

• Check the purification protein.
• Sample
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 10

None

September 11

Dialysis protein

• Dialysis and refolding for insoluble protein
• Sample
• Hv1a-his E1-E3
• Hv1a-lectin-his E1-E5
• Sf1a-his E1-E5
• Sf1a-lectin-his E1-E4
• OAIP-his E1-E3
• OAIP-lectin-his E1-E2

September 12

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 13

None

September 14

None

September 15

None

September 16

None

September 17

Dialysis protein

• Dialysis and refolding for insoluble protein
• Sample
• Hv1a-his E2-E3
• Hv1a-lectin-his E1-E6
• Sf1a-his E1-E4
• Sf1a-lectin-his E2-E4
• OAIP-his E2-E4
• OAIP-lectin-his E2-E3

September 18

None

September 19

None

September 20

None

September 21

None

September 22

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 23

Dialysis protein

• Remove urea
• Sample
• Hv1a-his E1-E3
• Hv1a-lectin-his E1-E6
• Sf1a-his E1-E4
• Sf1a-lectin-his E1-E6
• OAIP-his E1-E3
• OAIP-lectin-his E1-E6

SDS-PAGE

• Check the purification protein again
• Sample
• Hv1a-his
• Hv1a-lectin-his (no β-me)
• Sf1a-his
• Sf1a-lectin-his (no β-me)
• OAIP-his
• OAIP-lectin-his (no β-me)

Electrophoresis

• Electrophoresis of composite parts again
• Sample(8/30 PCR product)
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

September 24

Electrophoresis

• Electrophoresis of composite parts again
• Sample(8/30 PCR product)
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

September 25

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• Sf1a-lectin-his A^r
• OAIP-his A^r
• OAIP-lectin-his A^r

September 26

None

September 27

None

September 28

Protein quantification Braford method

• Sample
• Hv1a-his A^r
• Hv1a-lectin-his A^r
• Sf1a-his A^r
• OAIP-his A^r

September 29

None

September 30

None

October 1

None

October 2

None

October 3

None

October 4

None

October 5

Cultivation

• For larva test
• Sample
• Hv1a-GS linker-lectin A^r

October 6

None

October 7

None

October 8

Protein quantification Braford method

• Sample
• HLG-1
• HLG-2
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his
• BL
• RG

Protein quantification Braford method

• Sample
• HLG-1
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

October 9

None

October 10

None

October 11

Protein quantification Braford method

• Sample
• HLG-1
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his
• RG

SDS-PAGE

• Check the purification protein.
• Sample
• Hv1a(sonication)
• Hv1a(purification)
• Sf1a(sonication)
• Sf1a(purification)
• OAIP(sonication)
• OAIP(purification)

Protein quantification Braford method

• Sample
• HLG
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

Protein quantification Braford method

• Sample
• HLG(purification)
• Hv1a-his(purification)
• Hv1a-lectin-his(purification)
• Sf1a-his(purification)
• Sf1a-lectin-his(purification)
• OAIP-his(purification)
• OAIP-lectin-his(purification)
• Hv1a-his(sonication)
• OAIP-his(sonication)
• OAIP-lectin-his(sonication)
• HGL-his(sonication)
• Hv1a-his
• OAIP-lectin-his

October 12

None

October 13

None

October 14

None

October 15

SDS-PAGE

• Check the purification protein.
• Sample
• Hv1a-his
• Hv1a-lectin-his
• Sf1a-his
• Sf1a-lectin-his
• OAIP-his
• OAIP-lectin-his

October 16

None

October 17

None

October 18

None

October 19

None