Since it was the first week of doing experiments, we focused on some of the paperwork and the groundwork for the experiments ahead.

Drafted the protocol for the suicide module, prepared the LB medium, and initiated the revival of the Pseudomonas aeruginosa PAO1 strain. 

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

Made some changes and improvements on our molecular biology experiments design， then enriched our modules further.

suicide module

Received pcith and FADD this week. Amplified the target genes fragment of commercially synthesized pcith and FADD  by PCR. Upon reviewing the outcomes of the gel electrophoresis, we observed that pcith is positive while FADD kept showing negativeresults. 

Recovered the pcith and stored it for further usage.

suicide module

Retried to PCR FADD a few times more, but very sadly we didn't get positive results in the end.

Redesigned some parts of the suicide module and replaced the FADD with prpo and noksok.

Carbonic Anhydrase production module

Received P-CA this week. Amplified the target genes fragment of commercially synthesized  P-CA  by PCR. Purified PCR products.

suicide module

Amplified the target gene fragment of commercially synthesized pcith and prp by PCR. Identified PCR products by agarose gel electrophoresis. Purified PCR products.

Conducted enzymic cutting, mini prep, maxi prep with pcith and prpo.

Ligated PCR products pcith 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. Purify PCR products.

Carbonic Anhydrase production module

Conducted the enzymatic cutting for R-CA and P-CA, both are the genes responsible for the carbonic anhydrase expression.

Recovered the gene sequences and stored them to be used, since we got good feedback from fellow electrophoresis.

cellulose production module

Received BscA-BscB. Amplified the target gene fragment of commercially synthesized BscA-BscB by PCR. Identified PCR products by agarose gel electrophoresis. Purified PCR products.

suicide module

Ligated PCR products pcith with linearized vector PAB1 by homologous recombination to obtain the vectors: PAB1-prpo, PAB1-pcith and PBBR-MCS2-prpo.

Transfer the following vectors separately into P. aeruginosa by heat shock transformation. Conduct colony PCR and Sanger sequencing: PAB1-prpo, PAB1-pcith and PBBR-MCS2-prpo.

Electronic transmission module

1.Extracted the nqrF plasmids by alkaline lysis and obtain the nqrF plasmids by double-digestion with endonucleases XabI and HindIII.

Degradation module

1.Amplified the target gene fragment of commercially synthesized CYP-vgb by PCR. Identified PCR products by agarose gel electrophoresis. Purified PCR products.

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

Carbonic Anhydrase production module

Transfer the following vectors separately into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing: PBBR-MCS2-R-CA and PBBR-MCS2-p class = "text-content-p"-CA.

cellulose production module

Transfer the following vectors separately into E. coli by heat shock transformation. Conduct colony PCR and Sanger sequencing: BscA-BscB-MCS2-DH5α.

Cultured the strains and waited for the cellulose to produce.

suicide module

Received popDH this week. Amplified the target gene fragment of commercially synthesized popDH by PCR. Identified PCR products by agarose gel electrophoresis. Purified PCR products.

Conducted enzymatic cutting, mini prep, maxi prep, ligation and colony PCR for popDH.

Electronic transmission module

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

2.Ligated PCR products with linearized vector PAB1 by homologous recombination to obtain the vectors PAB1-nqrF, PAB1-PiliA, PAB1-PRPO-nqrF, PAB1-pcith-nqrF, PAB1-PRPO-PiliA and PAB1-pcith-PiliA.

3.Transfered the following vectors separately into E. coli by heat shock transformation. Conducted colony PCR and Sanger sequencing: PAB1-PiliA, PAB1-PiliA-pcith, PAB1-prop-nqrF, PAB1-nqrF, PAB1-pcith-nqrF and PAB1-prpo-PiliA.

Degradation module

1.Amplified the target gene fragment of commercially synthesized AlkB-ADH by PCR. Identified PCR products by agarose gel electrophoresis. Purified PCR products.

2.Obtained the PEBP-PEase, AlkB-ADH and CYP-vgb plasmids by double-digestion with endonucleases.

3.Extracted the PMV-AlkB-ADH plasmids by alkaline lysis.

4.Ligated PCR products with linearized vector PAB1 by homologous recombination to obtain the vectors PAB1-PEase-PEBP, PAB1-PRPO-PEase-PEBP and PAB1-pcith-PEase-PEBP and ligated PCR products with linearized vector PBBR-MSC2 by homologous recombination to obtain the vector PBBR-MSC2-AlkB-CVP-vgb.

5.Transfered the following vectors separately into E. coli by heat shock transformation. Conducted colony PCR and Sanger sequencing: PAB1-PEBP-PEase, PAB1-pcith-PEBP-PEase and PAB1-prpo-PEBP-PEase.

Electronic transmission module

1.Amplified the target gene fragment of commercially synthesized NadK-NadM by PCR. Identified PCR products by agarose gel electrophoresis. Purified PCR products.

2.Obtained the NadK-NadM plasmids by double-digestion with endonucleases.

3.Extracted the PAB1-PiliA and PAB1-pcith-PiliA plasmids by alkaline lysis.

Degradation module

1.Amplified the target gene fragment of commercially synthesized pntA-pntB by PCR. Identified PCR products by agarose gel electrophoresis. Purified PCR products.

2.Obtained the pntA-pntB, PEBP-GFP, AlkB-ADH, CYP-vgb, PEBP-GFP and PEBP-PEase plasmids by double-digestion with endonucleases.

3.Extracted the PMV-PEBP-GFP plasmids by alkaline lysis.

4.Ligated PCR products with linearized vector PAB1 by homologous recombination to obtain the vector PEBP-pcith-PEBP-GFP.

5.Transfered the following vectors separately into E. coli by heat shock transformation. Conducteds colony PCR and Sanger sequencing: PBBR-MSC2-CVP-vgb-AlkB-ADH.

For the degradation validation test, this week we conducted experiments on the comparison group. Specifically, we engaged 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. We prepared PE in 500µm and 3µm sizes. For both sizes, there were tubes with PAO1 and one tube without bacteria. After reproducing the bacteria, disinfecting the PE with ethanol, we transferred the PAO1 into the inorganic salt medium premixed with PE, ensuring that in the next subsequent 10days, the PE would serve as the bacteria's exclusive carbon source.

This week, we did 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 utilized two different particle sizes of PE, specifically 500µm and 3µm.

The first group of degradation validation test was ready for examination. We meticulously isolated the PE particles from the inorganic salt medium, and subjected them to go through the drying process within the oven. For the 500µm PE, we measured their weight loss throughout the 10 days co-cultivation period compared to the blank tube without the PAO1. Furthermore, we ran the FTIR test on both 500µm and 3µm PE, gathering and analyzing the data to identify the characteristic peaks.

The second group of degradation validation tests was ready for examination. Same process as the 10 days' group, we took the 500µm PE particles in the 15 days group for weight measuring and both 500µm and 3µm for FTIR.

We also did observations using SEM for PE with and without PAO1 degradation, sizes ranging from 3µm, 500µm, to film.

This week we re-examined the of 3µm PE sample of the 15 days co-cultivation period using the FTIR, since the data analysis from last week proved to be somewhat perplexing.

In addition to the degradation validation test, we also conducted tests to assess the gene function. Through IPTG cultivation, we performed preliminary experiment to measure the fluorescence intensity of the gene PAB1-pcith, therefore evaluating its expression.

Based upon preliminary work conducted last week, we did the formal experiment of the PAB1-pRPO, and measured the fluorescence intensity.

To test the production of cellulose, we utilized Congo Red staining to dye the sediments formed in the tubes. Having successfully demonstrated the synthesis of cellulose, we then ran a quantitative analysis of the sample.

Additionally, we performed effect validation for the nqrf sequence's and assessed the NADH ratio for the prpo sequence. Furthermore, we co-cultivate 11 tubes of PE and Escherichia coli strains harboring the inserted plasmids.

This week we conducted the FTIR tests on the 30 days co-cultivation period PE particles and the produced cellulose sample.

In a parallel effort to verify the expression of the flagella control gene, we performed a staining procedure on the flagella, followed by dyeing the examination under the microscope.

Hydroxylated the surface of the polished glass carbon sheet with the prepared Piranha lotion;

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

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

2.The insulation properties of SLBs were characterized, but the characterization failed.The results were 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 demonstrated the good stability of SLBs.

4.Prepared 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 was further demonstrated by stereomicroscope and the sink and float experiment of glass carbon sheet;

2.The insulation properties of SLBs were characterized, but the characterization failed.Further assessment indicated that the results were 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) was successfully tested with lower sweeps using electrochemical workstations.