Molecular Experiment

July 14th - July 15th

• Inoculate E. coli DH5$\alpha$ with plasmids PETase-MHETase and aldA-fucO and culture overnight.
• Extract plasmids PETase-MHETase and aldA-fucO.

July 16th - July 17th

• PCR plasmids PETase-MHETase and aldA-fucO to obtain linearized target gene fragments and vector fragments.
• Verify by agarose gel electrophoresis and purify the fragments.

July 18th - July 19th

• Homologous recombination of aldA-fucO and PETase-MHETase(vector).
• Transform the plasmid (homologous recombination) to E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. We failed to connect these fragments.

July 20th - July 21st

• Repeat PCR the plasmids PETase-MHETase and aldA-fucO to obtain linearized target gene fragments and vector fragments.
• Verify by agarose gel electrophoresis and purify the recovered fragments.

July 22nd - July 23rd

• Perform homologous recombination of aldA-fucO and PETase-MHETase (vector).
• Transform the plasmid (homologous recombination) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. The plasmid pPeteg is successfully validated, and the strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20 °C.

Fig 1. The PCR identification result of plasmid PETase-MHETase-aldA-fucO

July 24th - July 25th

• Inoculate E. coli DH5$\alpha$ with plasmids tphA2A3BA1-pUC57 and pBBR1-CS2 (vector), and culture overnight.
• Extract plasmids tphA2A3BA1-pUC57 and pBBR1-CS2.

July 26th - July 27th

• Double enzyme digestion of tphA2A3BA1-pBBR1-CS2(vector).
• Perform double enzyme digestion on the plasmids tphA2A3BA1-pUC57 and pBBR1-CS2 (vector), and purify the tphA2A3BA1 and pBBR1-CS2 (vector) fragments.
• Ligate the tphA2A3BA1 and pBBR1-CS2 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. The plasmid tphA2A3BA1-pBBR1-CS2 is successfully validated, and the strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20 °C.

Fig 2. The PCR identification result of plasmid tphA2A3BA1-pBBR1-CS2

July 28th - July 29th

• Inoculate E. coli DH5$\alpha$ with plasmids tpaK-pUC57 and pBBR1-CS2 (vector), and culture overnight.
• Extract plasmids tpaK-pUC57 and pBBR1-CS2.

July 30th - July 31st

• Double enzyme digestion of tpaK-pBBR1-CS2.
• Perform double enzyme digestion on the plasmids tpaK-pUC57 and pBBR1-CS2 (vector), and purify the tpaK and pBBR1-CS2 (vector) fragments.
• Ligate the tpaK and pBBR1-CS2 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. The plasmid tpaK-pBBR1-CS2 is successfully validated, and the strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20°C.

Fig 3. The PCR identification result of plasmid tpaK-pBBR1-CS2

August 1st - August 2nd

• Inoculate E. coli DH5$\alpha$ with plasmids tphA2A3BA1-pUC57 and tpaK-pBBR1-CS2 (vector), and culture overnight.
• Extract plasmids tphA2A3BA1-pUC57 and tpaK-pBBR1-CS2.

August 3rd - August 4th

• Double enzyme digestion of plasmids tphA2A3BA1-pUC57 and tpaK-pBBR1-CS2.
• Perform double enzyme digestion on the plasmids tphA2A3BA1-pUC57 and tpaK-pBBR1-CS2 (vector). However, we found that the length of the band obtained by double enzyme digestion of tpaK-pBBR1-CS2 (vector) is too short.

We suspect that due to the wrong designing of our plasmid, the tpaK gene fragment contained the restriction enzyme sites that would cut the gene fragment tpak into two pieces while we’re performing double enzyme digestion on plasmid tpak-pBBR1CS-2, resulting in an excess of wrong fragments with identical cohesive ends, which would ultimately causes most ligation products to be incorrect.

August 5th

• Perform PCR on the plasmid tphA2A3BA1-pUC57 to obtain the gene fragment tphA2A3BA1 with restriction enzyme sites of two new restriction endonucleases on both ends of the gene fragment tphA2A3BA1. Verify by agarose gel electrophoresis and purify the recovered fragments.

August 6th - August 7th

• Use new restriction endonucleases to perform double enzyme digestion on the linearized tphA2A3BA1 fragment and tpaK-pBBR1-CS2 (vector), and purify the tphA2A3BA1 and tpaK-pBBR1-CS2 (vector) fragments.
• Ligate the linearized tphA2A3BA1 fragment and tpaK-pBBR1-CS2 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. We failed to link these fragments.

August 8th - August 9th

• Repeat the process with new restriction endonucleases to perform double enzyme digestion on the tphA2A3BA1 fragment and tpaK-pBBR1-CS2 (vector), and purify the tphA2A3BA1 and tpaK-pBBR1-CS2 (vector) fragments.
• Ligate the linearized tphA2A3BA1 fragment and tpaK-pBBR1-CS2 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. The plasmid pTerephthalate (tphA2A3BA1-tpaK-pBBR1-CS2) is successfully validated, and an 1:1 mixture of double distilled water and glycerol and keep it at -20°C.

Fig 4. The PCR identification result of pTerephthalate

August 10th - August 11th

• Inoculate E. coli DH5$\alpha$ with plasmids rhlA-rhlB-pUC57 and pVLT33 (vector), and culture overnight.
• Extract plasmids rhlA-rhlB-pUC57 and pVLT33.

August 12th - August 13th

• Double enzyme digestion of rhlA-rhlB and pVLT33 (vector).
• Perform double enzyme digestion and ligation of rhlA-rhlB and pVLT33 (vector).
• Perform double enzyme digestion on the plasmids rhlA-rhlB-pUC57 and pVLT33 (vector), and purify the rhlA-rhlB and pVLT33 (vector) fragments.
• Ligate the rhlA-rhlB and pVLT33 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. We failed to link these fragments.

We believe that the similar lengths of the rhlA-rhlB fragment and the pUC57 fragment make it difficult to clearly separate bands during electrophoresis, preventing precise purification of the rhlA-rhlB gene fragment.

August 14th

• Obtain the rhlA-rhlB gene fragment through PCR from the plasmid rhlA-rhlB-pUC57 and purify the recovered fragment.

August 15th - August 16th

• Perform double enzyme digestion on the purified rhlA-rhlB fragment obtained after PCR and the plasmid pVLT33 (vector) using restriction endonucleases, and purify the double-digested rhlA-rhlB and pVLT33 (vector) fragments.
• Ligate the rhlA-rhlB and pVLT33 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. We failed to link these fragments.

August 17th

• Obtain the rhlA-rhlB gene fragment through PCR from plasmid rhlA-rhlB-pUC57 and purify the recovered fragment.

August 18th - August 19th

• Perform double enzyme digestion on the purified rhlA-rhlB fragment and the plasmid pVLT33 (vector), and purify the double-digested rhlA-rhlB and pVLT33 (vector) fragments.
• Ligate the rhlA-rhlB and pVLT33 (vector).
• Transform the plasmid (ligation) into E. coli DH5α and culture overnight.
• Pick bacterial plaque , PCR bacterial fluid , and verify by agarose gel electrophoresis. The rhlA-rhlB-pVLT33 plasmid is successfully validated. The strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20 °C.

Fig 5. The PCR identification result of rhlA-rhlB-pVLT33

August 20th - August 21st

• Inoculate E. coli DH5$\alpha$ with plasmids phaZ-pUC57 and pVLT33 (vector), and culture overnight.
• Extract plasmids phaZ-pUC57 and pVLT33.

August 22nd - August 23rd

• Double enzyme digestion of phaZ-pVLT33.
• Perform double enzyme digestion and ligation of phaZ and pVLT33 (vector).
• Use restriction endonucleases to perform double enzyme digestion on the plasmids phaZ-pUC57 and pVLT33 (vector) and purify the phaZ and pVLT33 (vector) fragments.
• Ligate the phaZ and pVLT33 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. We failed to link these fragments.

August 24th - August 25th

• Repeat the double enzyme digestion on the plasmids phaZ-pUC57 and pVLT33 (vector) using restriction endonucleases, and purify the phaZ and pVLT33 (vector) fragments.
• Ligate phaZ and pVLT33 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis.

We failed to link these fragments. We suspect that the low activity and amplification efficiency of the pVLT33 (vector) plasmid resulted in low ligation efficiency between pVLT33 and phaZ. We decide to proceed the expression validation for other plasmids first, and continue to construct plasmid phaZ-pVLT33 if our experiment schedule is relatively free.

September 2nd - September 3rd

• Repeat the double enzyme digestion on the phaZ-pUC57 and pVLT33 (vector), and purify the phaZ and pVLT33 (vector) fragments.
• Ligate the phaZ and pVLT33 (vector).
• Transform the plasmid (ligation) into E. coli DH5$\alpha$ and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. The validation is successful. The strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20 °C.

Fig 6. The PCR identification result of phaZ-pVLT33

Expression Validation

Validation of the degradation efficiency of terephthalic acid.

August 28th - August 29th

• Inoculate E. coli DH5$\alpha$ pTerephthalate and E. coli DH5$\alpha$ pBBR1-CS2, and culture overnight.
• Extract plasmids pTerephthalate.

August 30th - August 31st

• Perform electroporation to transfer the plasmids pTerephthalate and pBBR1-CS2 into P. putida KT2440 and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. Both plasmids are verified successfully, and the strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20 °C.

September 1st - September 7th

• Configure M9 medium with terephthalic acid as its carbon source, then cultivate P. putida KT2440 pTerephthalate and P. putida KT2440 pBBR1-CS2. Determine the growth curve of P. putida KT2440 pTerephthalate and P. putida KT2440 pBBR1-CS2.

September 8th

• Detect the remaining terephthalic acid by HPLC and verify the degradation efficiency of terephthalic acid in P. putida KT2440 pTerephthalate and P. putida KT2440 pBBR1-CS2.

Ramnolipid Detection

August 28th - August 29th

• Inoculate E. coli DH5$\alpha$ rhlA-rhlB-pVLT33 and E. coli DH5$\alpha$ pVLT33, and culture overnight.
• Extract plasmids rhlA-rhlB-pVLT33 and pVLT33.

August 30th - August 31st

• Use electroporation to transfer plasmids rhlA-rhlB-pVLT33 and pVLT33 into P. putida KT2440 and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. Both plasmids are verified successfully, and the strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20 °C.

September 1st - September 5th

• Configure M9 culture medium with soybean reconciled oil as its carbon source to culture P. putida KT2440 rhlA-rhlB-pVLT33 and P. putida KT2440 pVLT33.

Prepare the plate for TCL of rhamnolipid.

September 6th

• Use anthrone-sulfuric acid method and conduct TCL on P. putida KT2440 rhlA-rhlB-pVLT33 suspension and P. putida KT2440 pVLT33 to test their production of rhamnolipid

PET Degradation Efficiency Validation

September 9th - September 10th

• Inoculate E. coli DH5$\alpha$ with plasmids pPeteg, and culture overnight.
• Extract plasmid pPeteg.

September 11th - September 12th

• Transfer the plasmid pPeteg into E. coli BL21 and culture overnight.
• Pick bacterial plaque, PCR bacterial fluid, and verify by agarose gel electrophoresis. The plasmid is verified successfully, and the strain is stored in an 1:1 mixture of double distilled water and glycerol and keep it at -20 °C.

September 13th - September 14th

• Expand the culture of E. coli BL21 pPeteg, and add IPTG during the logarithmic growth phase ($\mathrm{OD_{600}}$=0.6-0.8).

September 15th

• Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the culture medium, but no target band is observed. We suspect that PETase-MHETase is not excreted.

September 16th - September 17th

• Inoculate and expand the culture of E. coli BL21 pPeteg, and add IPTG during the logarithmic growth phase ($\mathrm{OD_{600}}$=0.6-0.8).

September 18th

• Nickel column affinity chromatography and SDS-PAGE gel electrophoresis were performed on the supernatant and precipitate of the cell crushing solution, and both show a bright target band.

September 19th - September 26th

• Concentrate the supernatant to prepare crude enzyme, and mix the crude enzyme with PET powder and incubate. Detect the PET degradation products by HPLC to verify the enzyme activity and PET degradation efficiency of PETase-MHETase.

Validation of the Utilization Efficiency of Ethylene Glycol

September 13th - September 19th

• Prepare M9 medium with ethylene glycol and glycerol as its carbon sources, and expand the cultures of E. coli BL21 pPeteg and E. coli BL21 pUC19. Determine the growth curve of the two kinds of bacterias.