Team:WFLA YK PAO/Notebook

WFLA_YK_PAO

NOTEBOOK

August

• Day2 8/7

1. LB preparation

2. Transfer Top10 E. coli with IsPETase/MHETase containing pET28a into culture medium

• Day3 8/8

1. Plasmid extraction

2. Enzyme cut extracted plasmids

3. Detect plasmids with gel electrophoresis

4. Transfer plasmids into BL21 E. coli

5. Transfer BL21 E. coli into culture medium

• Day4 8/9

1. Prepare seed broth

2. Prepare IPTG inducer

3. Transfer BL21 E. coli and seed broth into culture medium

4. Prepare for Buffer A and Buffer B

5. Test for optic density of the bacteria strain

6. Add inducer into the bacteria solution and induce the expression of IsPETase and MHETase

• Day5 8/10

1. Adjust the pH of buffers

2. Test for optic density from the induced bacteria solution

3. Centrifugate the bacteria solution and preserve the rest of the bacteria

4. LB preparation

5. Add seed broth and induce bacteria solution into the shaking incubator

6. Test for optic density

7. Add IPTG inducer and put the solution in shaking incubator

8. Centrifugate to separate the bacteria

9. Break bacteria cell wall with ultrasound and preserve the proteins

10. Use nickle column purification to purify the expressed IsPETase proteins

• Day6 8/12

1. Use nickle column purification to purify the expressed MHETase proteins

2. Break uninduced bacteria cell wall with ultrasound and preserve the proteins

3. Prepare for SDS-page gel electrophoresis

4. Dye the gel and analyze the results

5. Overlapping PCR

a. Run PCR on IsPETase gene

b. Run PCR on MHETase gene

c. Run overlapping PCR on the products from the previous two PCRs

• Day7 8/13

1. Agarose gel electrophoresis on the products from the overlapping PCR

2. Observe and analyze the gel results

3. Redo PCR for IsPETase and MHETase gene

4. Redo SDS-page gel electrophoresis

5. Dye the gel and analyze the results

6. Redo agarose gel electrophoresis on the products from the overlapping PCR

7. Recycle the IsPETase and MHETase gene segments from the gel

8. Redo overlapping PCR step iii. to connect the two genes

• Day8 8/14

1. Redo agarose gel electrophoresis on the products from overlapping PCR

2. Recycle the connected gene segments from the gel

3. Enzyme cut the recycled gene segments

4. Run agarose gel electrophoresis on the cut genes

5. Recycle the cut gene segments from the gel

6. Connect the cut gene segments with cut pET28a

7. Induce the plasmid into competent cells

8. Add culture medium and centrifugate to preserve the bacteria

9. Prepare seed broth

10. Test for enzyme activity

• Day9 8/15

1. Redo PCR to amplify the connected IsPETase-MHETase gene segment

2. Run agarose gel electrophoresis on the connected gene segments

3. Recycle the connected gene segments from the gel

4. Enzyme cut the connected gene segments

5. Run agarose gel electrophoresis on the cut gene segments

6. Recycle the cut gene segments from the gel

7. Connect the cut gene segments with cut pET28a

8. Transfer the seed broth from 8/14 into the culture medium

9. Centrifugate to preserve the bacteria

10. Culture the bacteria on a solid culture medium

• Day10 8/16

1. Tranformed the plasmid pET28a-IsPETase-MHETase into E. coli BL21 and culture

2. Prepare Inducer and nickel column affinity purification related buffer

• Day11 8/17

1. Colony PCR

2. Protein induction and purification

September

1.Repeat transformation

2.Pilot expression

3. Sample preparation for SDS PAGE

4. Run SDS PAGE for verifying the expression of our engineered strain

5. Extract MHETase protein