This week we focused on some of the paperwork and the groundwork for the experiments ahead. 

1. Draft the protocol for the suicide module, prepare the LB medium, and initiate the revival of the Pseudomonas aeruginosa PAO1 strain. 

2. Conduct a preliminary test of the heat shock transformation technique on the PAO1, which unfortunately, whose results turned out to be negative.

Make some changes and improvements on our molecular biology experiments design. Enrich our modules further.

Suicide module

1. Receive the genes P_citH and FADD.  

2. Amplify the target genes fragment of commercially synthesized P_citH and FADD by PCR. Upon reviewing the outcomes of the gel electrophoresis, we observe that P_citH is positive while FADD keep showing negative results. 

3. Purify PCR products. Recover the P_citH and store it for further usage.

Suicide module

1. Retry to amplify the target genes fragment of commercially synthesized FADD  by PCR, but the result stays negative.

2. Redesign some parts of the suicide module and replace the P_citH with P_opdH.

Suicide module

1. Extract the pBBR1MCS2 plasmids by alkaline lysis.

2. Obtain the XbaI & KpnI, XbaI & SalI and KpnI & BaHI plasmids by double-digestion with endonucleases XbaI and NotI.

Carbonic Anhydrase production module

1. Receive the gene PAO102 this week.

2. Amplify the target genes fragment of commercially synthesized PAO102 by PCR.Identify PCR products by agarose gel electrophoresis.

3. Purify PCR products.

Suicide module

1. Amplify the target gene fragment of commercially synthesized P_opdH and pRPO by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

3. Conduct enzymic cutting, mini prep, maxi prep with P_opdH and pRPO.

4. Ligate PCR products P_opdH and pRPO with linearized vector pAB1 by homologous recombination.

Electronic transmission module

1. Amplify the target gene fragment of commercially synthesized nqrf by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

Carbonic Anhydrase production module

1. Conduct the enzymatic cutting for acaP and PAO102.

2. Recover the gene sequences and store them to be used.

Cellulose production module

1. Receive bscA-bscB.

2. Amplify the target gene fragment of commercially synthesized bscA-bscB by PCR. Identify PCR products by agarose gel electrophoresis.

3. Purify PCR products.

Suicide module

1. Ligate PCR products P_opdH with linearized vector pAB1 by homologous recombination to obtain the vectors: pAB1-pRPO, pAB1-P_opdH and pBBR1MCS2-pRPO.

2. Transfer the following vectors separately into P. aeruginosa by heat shock transformation. Conduct colony PCR and Sanger sequencing: pAB1-pRPO, pAB1-P_opdH and pBBR1MCS2-pRPO.

Electronic transmission module

Extract the nqrf plasmids by alkaline lysis and obtain the nqrf plasmids by double-digestion with endonucleases XabI and HindIII.

Degradation module

1. Amplify the target gene fragment of commercially synthesized cypY96F-vgb by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

3. Extract the vgb plasmids by alkaline lysis and obtain the vgb plasmids by double-digestion with endonucleases XabI and NotI.

Carbonic Anhydrase production module

1. Transfer the following vectors separately into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing: pBBR1MCS2-acaP and pBBR1MCS2-PAO102.

Cellulose production module

1. Transfer the following vectors separately into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing: bscA-bscB-pBBRMCS2-DH5α.

2. Culture the strains and wait for the cellulose to produce.

Suicide module

1. Receive hok/sok this week.

2. Amplify the target gene fragment of commercially synthesized hok/sokby PCR. But the result is negative.

Electronic transmission module

1. Amplify the target gene fragment of commercially synthesized pntA-pntB by PCR. However, the agarose gel electrophoresis shows that we are not able to amplify the correct PCR products.

2. Ligate PCR products with linearized vector pAB1 by homologous recombination to obtain the vectors pAB1-nqrf, pAB1-pilA, pAB1-pRPO-nqrf, pAB1-P_citH-nqrf, PAB1-pRPO-pilA and pAB1-P_citH-pilA.

3. Transfer the following vectors separately into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing: pAB1-nqrf, pAB1-pilA, pAB1-pRPO-nqrf, pAB1-P_citH-nqrf, PAB1-pRPO-pilA and pAB1-P_citH-pilA.

Degradation module

1. Amplify the target gene fragment of commercially synthesized alkB2-adhA by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

3. Obtain the PEBP-PEase, alkB2-adhA and cypY96F-vgb plasmids by double-digestion with endonucleases.

4. Extract the pMV-alkB2-adhA plasmids by alkaline lysis.

5. Ligate PCR products with linearized vector pAB1 by homologous recombination to obtain the vectors pAB1-PEase-PEBP, pAB1-pRPO-PEase-PEBP and pAB1-P_citH-PEase-PEBP.

6. Ligate PCR products with linearized vector pBBR1MCS2 by homologous recombination to obtain the vector pBBR1MCS2-alkB2-cypY96F-vgb.

7. Transfer the following vectors separately into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing: pAB1-PEBP-PEase, pAB1-P_citH-PEBP-PEase and pAB1-pRPO-PEBP-PEase.

Electronic transmission module

1. Amplify the target gene fragment of commercially synthesized nadK-nadM by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

2. Obtain the nadK-nadM plasmids by double-digestion with endonucleases.

3. Extract the pAB1-pilA and pAB1-P_citH-pilA plasmids by alkaline lysis.

Degradation module

1. Amplify the target gene fragment of commercially synthesized pntA-pntB by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

3. Obtain the pntA-pntB, PEBP-GFP, alkB2-adhA, cypY96F-vgb and PEBP-PEase plasmids by double-digestion with endonucleases.

4. Extract the pMV-PEBP-GFP plasmids by alkaline lysis.

5. Ligate PCR products with linearized vector PAB1 by homologous recombination to obtain the vector PEBP-P_citH-PEBP-GFP.

6. Transfer the following vectors separately into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing: pBBR1MCS2-cypY96F-vgb-alkB2-adhA.

Cellulose production module

1. Extract the bcsA-bcsB plasmids by alkaline lysis.

2. Obtain the pMV-bcsA-bcsB plasmids by double-digestion with endonucleases.

Suicide module

1. Extract the pMV-hok/sok plasmids by alkaline lysis.

2. Obtain the hok/sok by double-digestion with endonucleases.

3. Ligate hok/sok with linearized vector pAB1 by T₄ ligation enzyme to obtain the vector pAB1-hok/sok but fail.

Electronic transmission module

1. Extract the pAB1-nadK-nadM-pntA-pntB, pMV-nadK-nadM, pMV-pntA-pntB, and pBBR1MCS2-nadK-nadM-pntA-pntB plasmids by alkaline lysis.

2. Obtain the pBBR1MCS2-nadK-nadM-pntA-pntB plasmids by double-digestion with endonucleases.

Degradation module

1. Obtain the pntA-pntB, PEBP-GFP, alkB2-adhA, cypY96F-vgb, PEBP-GFP, and PEBP-PEase plasmids by double-digestion with endonucleases.

2. Extract the pMV-PEBP-GFP and pBBR1MCS2-cypY96F-alkB2 plasmids by alkaline lysis.

Promoters

1. The pS and Pbla promoter genes arrive. The pAB1-pS and pAB1-Pbla are constructed using the pS and Pbla genes and then transformed into E.coli DH5α.

Cellulose production module

1. Ligate pBBR1MCS2-bcsA-bcsB with linearized vector pAB1 by homologous recombination to obtain the vector pBBR1MCS2-bcsA-bcsB.

2. Amplify the target gene fragment of commercially synthesized bcsA-bcsB by PCR. Identify PCR products by agarose gel electrophoresis.

3. Purify PCR products.

Suicide module

1. Extract the pMV-hok/sok from bacteria and conduct double-digestion with endonucleases to gain hok/sok.

2. PCR to get hok/sok and P_opdH with homologous arms.

3. Ligate hok/sok and P_opdH separately with linearized vector pAB1 by homologous recombination to obtain the vector pAB1-hok/sok and pAB1-P_opdH-GFP.

Electronic transmission module

1. Amplify the target gene fragment of commercially synthesized nadK-nadM by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

3. Extract the pMV, pMV-nqrf, pMV-pntA-pntB, and pMV-nadK-nadM plasmids by alkaline lysis.

4. Ligate pS-pilA and Pbla-nqrf with linearized vector pAB1 by homologous recombination to obtain the vector pAB1-pS-pilA and pAB1-Pbla-nqrf.

5. Obtain the pBBR1MCS2 plasmids by double-digestion with endonucleases.

Degradation module

1. Extract the pMV, pMV-PEBP-PEase, pMV-PEBP-GFP, pMV-alkB2-adhA, and pMV-cypY96F-vgb by alkaline lysis.

2. Ligate PCR products with linearized vector pBBR1MCS2 by homologous recombination to obtain the vector pBBR1MCS2-cypY96F-vgb-alkB2-adhA, and ligate PCR products with linearized vector pAB1 by homologous recombination to obtain the vector pAB1-cypY96F-vgb, pAB1-alkB2-adhA, and pAB1-PEBP-PEase.

3. Amplify the target gene fragment of commercially synthesized cypY96F-vgb by PCR. Identify PCR products by agarose gel electrophoresis.

4. Purify PCR products.

Cellulose production module

1. Conduct colony PCR and Sanger sequencing: pBBR1MCS2-bcsA-bcsB.

2. Ligate PCR product bcsA-bcsB with linearized vector pBBR1MCS2 by homologous recombination.

Suicide module

1. Conduct colony PCR and Sanger sequencing: pAB1-hok/sok.

Electronic transmission module

1. Amplify the target gene fragment of commercially synthesized pMV-nqrf by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

3. Obtain the pS-nqrf plasmids by double-digestion with endonucleases.

4. Extract the pAB1-pilA plasmids by alkaline lysis.

5. Transfer the pAB1-Poprl-pilA vector into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing.

6. Amplify the target gene fragment of commercially synthesized pMV-nadM-nadK and pMV-pntA-pntB by PCR. Identify PCR products by agarose gel electrophoresis.

Suicide module

1. Transfer the pAB1-hok/sok A vector into Pseudomonas aeruginosa PAO1 by electronic transformation.

Degradation module

1. Amplify the target gene fragment of commercially synthesized alkB2-adhA-Ty-pBBR1MCS2, alkB2-adhA-Ty-pAB1 by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

3. Ligate PCR product pAB1-cypY96F with linearized vector pAB1-pS by homologous recombination.

4. Ligate pS-PEBP-GFP and pS-PEBP-PEase with linearized vector pAB1 by homologous recombination to obtain the vector pAB1-pS-PEBP-GFP and pAB1-pS-PEBP-PEase.

5. Extract the plasmid pAB1-alkB2.

Cellulose production module

1. Amplify the target gene fragment of commercially synthesized bcsA-bcsB by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

Electronic transmission module

1. Transfer the pAB1-pS-nqrf vector into E. coli by heat shock transformation.

2. Conduct colony PCR and Sanger sequencing on pilA-pAB1, pAB1-nqrf, pAB1-pS-nqrf, and pAB1-pS-nqrf.

3. Testify the plasmid nqrf.

4. Amplify the target gene fragment of commercially synthesized pMV-nadM-nadK by PCR. Identify PCR products by agarose gel electrophoresis.

5. Extract the pBBR1MCS2 plasmids by alkaline lysis.

6. Obtain the pBBR1MCS2 plasmids by double-digestion with endonucleases.

7. Ligate nadM-nadK and pntA-pntB with linearized vector pBBR1MCS2 by homologous recombination.

8. Transfer the pBBR1MCS2-nadM-nadK-pntA-pntB vector into E. coli by heat shock transformation.

9. Conduct colony PCR on pBBR1MCS2-nadM-nadK-pntA-pntB.

Degradation module

1. Amplify the target gene fragment of commercially synthesized PEBP-GFP, PEBP-PEase by PCR. Identify PCR products by agarose gel electrophoresis.

2. Transfer the PEBP-PEase vector into P. aeruginosa PAO1 by electronic transformation.

3. Conduct colony PCR and Sanger sequencing on pAB1-cypY96F-vgb, PEBP-PEase, PEBP-GFP, alkB2-vgb-PAO1.

4. PAO1 transformed with the plasmids pAB1-alkB2-adhA and pAB1-cypY96F-vgb, as well as BL21 transformed with the plasmids pAB1-alkB2-adhA and pAB1-cypY96F-vgb, are co-cultivated with plastic, and IPTG is added to the medium for induction.

5. Using BL21 transformed with pAB1-alkB2-adhA and pAB1-cypY96F-vgb, as well as PAO1, we lyse the cells, extract proteins, and perform SDS-PAGE.

Carbonic Anhydrase production module

1. Ligate PCR product PAO102 with linearized vector pAB1-pS by homologous recombination.

2. Obtain the plasmid pAB1-pS-PAO102 by homologous recombination.

3. Obtain the pAB1-pS plasmids by double-digestion with endonucleases.

4. Conduct colony PCR on DH5α-pAB1-pS-PAO102.

Electronic transmission module

1. Ligate nadM-nadK with pntA-pntB by homologous recombination.

2. Amplify the target gene fragment of nadM-nadK-pntA-pntB by PCR. Identify PCR products by agarose gel electrophoresis.

3. Ligate nadM-nadK-pntA-pntB with linearized vector pBBR1MCS2 by homologous recombination.

4. Transfer the pBBR1MCS2-nadM-nadK-pntA-pntB vector into E. coli by heat shock transformation.

5. Conduct colony PCR on pBBR1MCS2-nadM-nadK-pntA-pntB.

Cellulose production module

1. Obtain the plasmid pBBR1MCS2-bcsA-bcsB by homologous recombination.

2. For PAO1 with the plasmids pAB1-nqrf, pAB1-pilA, and pBBR1MCS2-bcsA-bcsB, as well as for untransformed PAO1, we lyse the cells and extract proteins for SDS-PAGE analysis.

Degradation module

1. Obtain the PEBP-GFP plasmids by double-digestion with endonucleases.

2. Conduct colony PCR on PAO1-PEBP-GFP.

3. For PAO1 with the plasmids pAB1-PEBP-PEase, pAB1-PEBP-GFP, as well as for untransformed PAO1 and BL21 transformed with plasmids pAB1-PEBP-PEase and pAB1-PEBP-GFP, we lyse the cells and extract proteins for SDS-PAGE analysis.

For the degradation validation test, this week we conduct experiments on the comparison group. Specifically, we engage in the co-cultivation of Polyethylene and non-modified Pseudomonas aeruginosa PAO1 over a period of 10 days. Basically, we want to look at the degradation efficiency difference between the wild type of bacteria and the ones after gene editing.

1. Prepare PE in 500μm and 3μm sizes. For both sizes, there are tubes with PAO1 and one tube without bacteria.

2. Reproduce the bacteria, disinfect the PE with ethanol, and transfer the PAO1 into the inorganic salt medium premixed with PE.

3. Co-cultivate for 10 days.

This week, we run other two groups of the degradation validation test, with one group of 15 days of co-cultivation and another for 30 days. For both groups, we utilize two different particle sizes of PE, specifically 500μm and 3μm.

1. Prepare PE in 500μm and 3μm sizes. For both sizes, there are tubes with PAO1 and one tube without bacteria.

2. Reproduce the bacteria, disinfect the PE with ethanol, and transfer PAO1 into the inorganic salt medium premixed with PE.

3. Co-cultivate. One group for 15 days and another group for 30 days.

1. Isolate the 10 days co-cultivate PE particles from the inorganic salt medium.

2. Dry the PE particles in the oven.

3. Measure their weight loss for 500μm PE throughout the 10 days co-cultivation period compared to the blank tube without the PAO1.

4. Conduct the FTIR test on both 500μm and 3μm PE.

1. Isolate the 15 days co-cultivate PE particles from the inorganic salt medium

2. Dry the PE particles.

3. Measure the weight loss of the 500μm PE throughout the 15 days co-cultivation period compared to the blank tube without the PAO1.

4. Conduct the FTIR test on both 500μm and 3μm PE.

5. Conduct SEM on PE films and particles (3μm, 500μm) with and without PAO1 degradation.

1. Conduct the FTIR test on both 500μm and 3μm PE again, because last week's data was not clear.

2. Conduct IPTG cultivation and perform preliminary experiment to measure the fluorescence intensity of the gene PAB1-P_cith, to evaluating its expression.

1. Conduct the formal experiment of the pAB1-pr based on preliminary work conducted last week, and measure the fluorescence intensity.

2. Utilize Congo Red staining to dye the sediments formed in the tubes. Having successfully demonstrated the synthesis of cellulose, we then run a quantitative analysis of the sample.

3. We perform effect validation for the nqrf sequence's and assess the NADH ratio for the pRPO sequence.

4. Co-cultivate 11 tubes of PE and E. coli strains harboring the inserted plasmids.

1. Conduct the FTIR tests on the 30 days co-cultivation period PE particles and the produced cellulose sample.

2. To verify the expression of the flagella control gene, we perform a staining procedure on the flagella, followed by dyeing the examination under the microscope.

1. Conduct the FTIR tests on the 10-day co-cultivation period PE particles with the vector-transferred E. coli.

2. Conduct generation passing to bacteria with and without the hok/sok system in antigen-free LB broth. Streak corresponding groups on the plates with Amp to verify plasmid loss in bacteria and conduct mortality statistics.

3. The pS and Pbla promoters are verified to function as constitutive promoters that continuously drive expression in PAO1.

1. Co-cultivate the 500μm PE particles with PAO1 transferred in with the vectors pAB1-alkB2-adhA and pAB1-cypY96F-vgb.

2. Continue the generation passing experiment of bacteria with and without hok/sok.

1. Cellulose is extracted from 30 mL of the control group bacterial fluid and 30 mL of the experimental group bacterial fluid.

2. Conduct FTIR and SEM examination on the co-cultivated samples.

3. The NADH/NADH⁺ assay kit is used to verify the function of Nqr.

4. The OD600 growth curve of Rhodopseudomonas palustris CGA009 and P. aeruginosa PAO1 co-incubation is measured.

5. Cultivate bacteria with pAB1-hok/sok and pAB1-P_opdH-GFP. Divide bacteria with pAB1-hok/sok into groups with and without IPTG (0.6 mM) and observe the growth based on OD600 detection by enzyme labeling apparatus. Divide bacteria with pAB1-PopdH-GFP into groups with and without citrate (5 mM) and observe the fluorescence intensity by enzyme labeling apparatus. Conduct statistics.

6. Divide bacteria with pAB1-hok/sok into groups with and without IPTG (0.6 mM) and observe the growth based on OD600 detection by enzyme labeling apparatus. Divide bacteria with pAB1-P_opdH-GFP into groups with and without citrate (5 mM) and observe the fluorescence intensity by enzyme labeling apparatus. Conduct statistics.

1. Dissolve 20mg of DOPC and 0.2mg of Texas Red ^@DHPE in chloroform. Take 8mL of the solution, replace the organic phase with the aqueous phase by blowing nitrogen into it, and store the mixture in a 4°C refrigerator overnight.

2. Use an ultrasonic crusher to process yesterday's solution until it is clarified, obtaining a small unilamellar vesicle (SUV) solution. Centrifuge the SUV solution at 12,000xg for 8 hours, then remove the supernatant and store it in a refrigerator at 4°C.

1. Hydroxylate the surface of the polished glass carbon sheet with the prepared Piranha lotion.

2. SLBs solution is prepared, and uniform SLBs are successfully detected on the surface of the glass carbon sheet by using fluorescence microscope.

1. Prepare electrolyte (0.5mol/L sodium chloride + 0.05 mmol/L potassium ferricyanide ).

2. The insulation properties of SLBs are characterized, but the characterization fails. The results are likely affected by the reference parameter setting of the electrochemical workstation or electrolyte ratio.

3. The fluorescence characterization of the glass carbon sheet after use in the electrolyte demonstrates the good stability of SLBs.

4. Prepare two kinds of electrolyte (0.5mol/L sodium chloride + 0.05 mmol/L potassium ferricyanide and 0.5mol/L sodium chloride + 0.05 mmol/L potassium ferricyanide).

1. The hydroxylation of the surface of glass carbon sheets using Piranha wash is further demonstrated by stereomicroscope and the sink and float experiment of glass carbon sheet;

2. The insulation properties of SLBs are characterized, but the characterization fails. Further assessment indicate that the results are affected by the reference parameter setting of the electrochemical workstation, instead of electrolyte ratio;

3. The blank background of the three-electrode system (EIS \ CV) is successfully tested with lower sweeps using electrochemical workstations.

1. Complete the construction of the 3D solid model of the electron transfer chain detection system.

2. The construction of the 3D solid model of the peristaltic pump for microplastics detection has been completed.

1. Construct the conductive part of the electron transfer chain detection system.

2. Assemble the electron transfer chain detection system.

1. The construction of the 3D solid model of the central sample pool for microplastic content detection has been completed.

2. Assemble the peristaltic pump and measure the output pulse and central shaft speed.

1. Hydroxylate the surface of the polished glassy carbon sheet with the prepared Piranha lotion.

2. Dissolve 12 mg of DOPC, 0.12 mg of Texas Red ^@DHPE, and 2.4 mg of DGS-NTA (Ni) in 10 mL of chloroform. Replace the organic phase with the aqueous phase by nitrogen blowing, and place it in a refrigerator at 4°C overnight.

3. Use an ultrasonic homogenizer to clarify the solution from yesterday. Centrifuge the SUV solution at 12,000xg for 8 hours, then remove the supernatant and store it in a refrigerator at 4°C.

1. MasC and PEBP are loaded onto functional SLBs and characterized using fluorescence.

2. Membranes are characterized using EIS and CV with an electrochemical workstation.

3. Microbial Fuel Cell Test (pAB1-pS-nqrf, pAB1-pS-pilA).