iGEM 2024 — Team bnuzh-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

Notebook

![](https://static.igem.wiki/teams/5291/images/page-header4.svg)
![](https://static.igem.wiki/teams/5291/images/overview/wetlab2.jpg)

Notebook

Molecular Experiments

June

July

August

September

Validation Experiments

July

August

September

Hardware Experiments

July

August

September

Molecular Experiments

June

Week 1 | 6.24~6.30

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.

July

Week 2 | 7.1~7.7

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

Week 3 | 7.8~7.14

**Suicide module**

1. Receive the genes PcitH and *FADD*.

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

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

Week 4 | 7.15~7.21

**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 PcitH with PopdH.

Week 5 | 7.22~7.28

**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*.

Week 6 | 7.29~8.4

**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 PopdH and pRPO by PCR. Identify PCR products by agarose gel electrophoresis.

2. Purify PCR products.

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

4. Ligate PCR products PopdH 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.

August

Week 7 | 8.5~8.11

**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 PopdH with linearized vector pAB1 by homologous recombination to obtain the vectors: pAB1-pRPO, pAB1-PopdH 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-PopdH 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.*

Week 8 | 8.12~8.18

**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/sok*by 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-PcitH-*nqrf*, PAB1-pRPO-*pilA* and pAB1-PcitH-*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-PcitH-*nqrf*, PAB1-pRPO-*pilA* and pAB1-PcitH-*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-PcitH-*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-PcitH-*PEBP-PEase* and pAB1-pRPO-*PEBP-PEase*.

Week 9 | 8.19~8.25

**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-PcitH-*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*-PcitH-*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.*

Week 10 | 8.26~9.1

**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.

September

Week 11 | 9.2~9.8

**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 PopdH with homologous arms.

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

Week 12 | 9.9~9.15

**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*.

Week 13 | 9.16~9.22

**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.

Week 14 | 9.23~9.29

**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.

Validation Experiments

July

Week 2 | 7.1~7.7

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.

Week 3 | 7.8~7.14

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.

Week 4 | 7.15~7.21

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.

Week 6 | 7.29~8.4

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.

August

Week 7 | 8.5~8.11

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-Pcith, to evaluating its expression.

Week 8 | 8.12~8.18

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.

Week 9 | 8.19~8.24

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.

Week 10 | 8.26~9.1

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.

September

Week 13 | 9.16~9.22

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*.

Week 14 | 9.23~9.29

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-PopdH-*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-PopdH-*GFP* into groups with and without citrate (5 mM) and observe the fluorescence intensity by enzyme labeling apparatus. Conduct statistics.

Hardware Experiments

July

Week4 | 7.15~7.21

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.

Week5 | 7.22~7.28

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.

Week 6 | 7.29~8.4

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).

August

Week 7 | 8.5~8.11

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.

Week 8 | 8.12~8.18

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

Week 9 | 8.19~8.25

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

Week 10 | 8.26~9.1

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

2. Assemble the electron transfer chain detection system.

September

Week 11 | 9.2~9.8

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

Week 12 | 9.9~9.15

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

Week 13 | 9.16~9.22

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.

Week 14 | 9.23~9.29

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*).

{% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your
project by going through at least one iteration of the engineering design cycle.
This achievement should be distinct from your Contribution for Bronze.{%
endblock %} {% block page\_content %} {# 初始化环形导航栏内容 #} {% set labels =
[ {"name": "Design", "x": 61, "y": 19, "r": 1}, {"name": "Build", "x": 63, "y":
68, "r": -1}, {"name": "Test", "x": 14, "y": 67, "r": 1}, {"name": "Learn", "x":
14, "y": 16, "r": -1} ] %} {# 定义生成单个组的宏，每次使用都依据全局计数器#}
{% macro generate\_circle\_container(offset) %} {% set offset\_img = (offset-1) \* 5
+ 1%} {% set offset\_item = (offset-1) \* 4 + 1%}

Click
the
cycle!

{% for i in range(4) %} {% set idx = i + offset\_img %}
![{{ labels[i].name|lower }}](https://static.igem.wiki/teams/5291/images/engineering/{{ labels[i].name|lower }}-activation.svg)
{% endfor %}
![base](https://static.igem.wiki/teams/5291/images/engineering/no-activation.svg)

{% for i in range(4) %} {% set label = labels[i] %}

{{ label.name }}

{% endfor %}

{% endmacro %}

Engineering

![](https://static.igem.wiki/teams/5291/images/page-header4.svg)
![](https://static.igem.wiki/teams/5291/images/overview/wetlab2.jpg)

Degradation

EET & CO2 Transfer

Carbon Fixation
Safety

Engineering

Degradation Module

{{ generate\_circle\_container(1) }}

Cycle 1:Predicting PEBP Sequence

Cycle 2:Modifying the Promoter

Cycle 3:Expressing PE Binding Peptide-PEase Fusion Protein

Cycle 4:Improving the Downstream Assimilation and Mineralization
pathways

References

The whole process of biodegradation can be classified into four
steps: Biodeterioration, Degradation, Assimilation and
Mineralization. Degradation ability of engineered
*Pseudomonas aeruginosa* PAO1 will be gradually strengthened
according to the above four steps.

Cycle 1: Predicting PEBP Sequence

Design

According to the needs of the experiment, we need to construct a
predictor to determine whether a peptide sequence can bind to
PE. After the preliminary literature search, there are 174
pieces of related data collected. Due to the small amount of
data, we first performed machine learning training; then we
performed special processing and tried to introduce neural
networks.

Build

Firstly, the original data is used to train the SVM model; then,
the data is increased by 1:10 to get nearly two thousand pieces
of data. Then, the trained SVM is used to sift the augmented
data according to specific criteria to get a new data set; to
dig deeper into the sequence information, the sequence is
transformed into a structural graph, the features are extracted
using graph neural network (GNN) and location specific scoring
matrix, and the attention module is introduced to perform
further learning to get a predictor.

Test

The above dataset is divided into training and test sets,
experiments are conducted using the built model and
corresponding experimental metrics such as accuracy, recall and
F1 score are measured.

Learn

Finally, the test results are further analysed and the model
parameters are adjusted and optimised.

Cycle 2: Modifying the Promoter

Design

The initial step of constructing a plasmid is finding an
appropriate plasmid backbone. We chose pAB1, a broad host range
plasmid for IPTG-inducible expression of EGFP, as our basic
plasmid backbone for expressing protein in *P. aeruginosa*.
After ordering the plasmid, we found that RBS sequence will be
cut off if changes the GFP sequencing to our gene of interest.
And if we want to release our engineered bacteria to the soil, a
constitutive expression promoter is needed. Replacing the
promoter is an urgent need.

Build

We selected 4 promoters that were indicated in the paper that
could be expressed in *P. aeruginosa*, pRPO, P*bla*, P*oprL*
and pS.Then linked the promoter sequence into the sequence by
enzyme cleavage of NsiI and XbaI on pAB1.

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/figure1.png)

Figure 1 Construction of pAB1-pS plasmid

Test

The pAB1 plasmids with new promoter were transferred into the
DH5α strain, and EGFP was successfully expressed.

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/figure2.jpg)

Figure 2 The DH5α strain with pAB1-pS-*egfp* and
pAB1-PoprL-*egfp* plasmids can exsert fluorescence

Learn

When synthesizing short promoter fragments, we used the
annealing extension method of two complementary single strand
DNA for rapid synthesis. We selected the cleavage sites of NsiI
and XbaI for enzyme digestion and ligation operation, replacing
the lac operator and ptrc by our aiming promoters. Initially, we
tried to use pRPO as a constitutive promoter, but the pRPO
promoter could drive the expression of gfp gene in
*E.coli* DH5α strain, but could not drive the expression of
the gene in *P. aeruginosa* PAO1. Therefore, we later found
pS, P*oprL* and P*bla* promoters for testing, and found that gfp
driven by all three of these promoters could be successfully
expressed in PAO1 strain. Given that the pS promoter strain
expressed the strongest and most stable fluorescence intensity,
we ended up using the pS promoter as our constitutive promoter.

Cycle 3: Expressing PE Binding Peptide-PEase Fusion Protein

Design

After the microorganisms approaching to the microplastics, they
can begin the biodeterioration step. Therefore, we intend to
construct a fusion protein of PEBP and PE degrading enzymes to
enhance the biodeterioration ability of our engineered bacteria.
Different kinds of PE degrading enszymes play a role in
different situations, so it is crucial to select a suitable PE
degrading enzyme, and PEase from wax worm saliva is a suitable
candidate. We couple PEBP to PEase. Signal6.0 was used to
predict a signal peptide of Esterase-A from
*P. aeruginosa* and bind to the N-terminal of PEBP. The
membrane binding domain is predicted using software and is
attached to the C end of the PEase. In this way, we have
designed a whole PE binding and deteriorate module.

![img](https://static.igem.wiki/teams/5291/images/engineering/f3.png)

Figure 3 Gene line diagram of pAB1-*PEBP-PEase*

![img](https://static.igem.wiki/teams/5291/images/engineering/f4.png)

Figure 4 Gene line diagram of pAB1-*PEBP-GFP*

Build

We ordered the complete PE adsorption-depolymerization module
sequence from a company and cloned it into the pAB1 vector using
restriction enzymes. Colony PCR and sequencing confirmed the
successful construction of our vector. We introduced an enzyme
cutting site between PEBP and PEase to facilitate the
replacement of different PEBP sequences. Additionally, we had the
company synthesize a sequence with PEBP fused to EGFP to assess
the ability of PEBP-expressing bacteria to bind to
microplastics.

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/figure-5.png)

Figure 5 Construction of pAB1-*PEBP-PEase* plasmid

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/figure-6.png)

Figure 6 Construction of pAB1-*PEBP-GFP* plasmid

Test

SDS page was used for gene expression detection, and the correct
bands of pAB1-*PEBP-GFP* were obtained, indicating that the
protein was indeed correctly expressed in the bacteria. However,
we didn't obtain correct band of pAB1-*PEBP-PEase* plasmid, so for
this plasmid we tested it by modeling method.

The PEBP-GFP protein was purified and extracted. From the
experimental results, we can clearly see that the fluorescence
intensity of PEBP-GFP co-incubated microplastics is obviously
higher than GFP co-incubated microplastics and blank control
group, indicating that PEBP does play a role in adsorption.

For PEase, we tested its effect through moduling. We conducted
structure predicting of PEBP-PEase. The predicting module
clearly displayed a membrane spanning domain and an enzyme
domain, which we exactly expected our bacteria to produce. After
that molecular docking was conducted to study the interactions
between the PEase enzyme and both large and small alkane
molecules. The result indicating that PEase is able to
deteriorate PE plastic.

Learn

In this cycle we successfully constructed pAB1-*PEBP-PEase*
and pAB1-*PEBP-GFP* plasmids, the effects of PEBP were
verified by co-incubation, and PEase was verified by modeling
methods.

Cycle 4: Improving the Downstream Assimilation and Mineralization
Pathways.

Design

Microplastics are depolymerized into smaller plastic molecules,
which then need to be assimilated by microorganisms and further
depolymerized within the cells. In the assimilation step,
short-chain alkanes can pass through the cell membrane on their
own, while medium-short-chain alkanes can enter the cell via
AlkB2. For the subsequent mineralization step, we introduced a
CYP enzyme with the CYPY96F mutation to efficiently hydroxylate
free short-chain alkanes. Medium-long-chain alkanes can be
hydroxylated during their entry through AlkB2. To improve the
hydroxylation efficiency of medium-long-chain alkanes, we
coupled the Rd45 auxiliary factor to AlkB2 to assist with
electron transfer. Given that ethanol dehydrogenation is a
crucial step in alkane chain mineralization, we also coupled
AdhA ethanol dehydrogenase, which can dehydrogenate long-chain
alcohols, to AlkB2 to facilitate downstream reactions.

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/fig-7.png)

Figure 7 Gene line diagram of pAB1-*alkB2-Rd45-adhA*
plasmid

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/fig-8.png)

Figure 8 Gene line diagram of pAB1-*cypY96F-vgb* plasmid

Build

We had the company synthesize fragments of the AlkB2-Rd45-AdhA
fusion protein and the CYP-VHb fragment, adding enzyme cutting
sites at both ends. We ultimately constructed the entire vector
using restriction cloning. We obtained the correct colony PCR
results and sequencing results, indicating that we successfully
constructed the vector.

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/figure-9.png)

Figure 9 Construction of pAB1-*alkB2-Rd45-adhA* plasmid

![img](https://static.igem.wiki/teams/5291/images/engineering/degradation/figure-10.png)

Figure 10 Construction of pAB1-*cypY96F-vgb* plasmid

Test

SDS page was used for gene expression detection, and the correct
bands were obtained, indicating that the protein was indeed
correctly expressed in the bacteria.

![img](https://static.igem.wiki/teams/5291/images/result/new/f6.png)

Figure 11(left) Electropherogram of AlkB2-Rd45-AdhA colony PCR
gel
Figure 12(right) Electropherogram of CYP96F-VHb colony
PCR gel

For Degradation module, we tested its effect through
co-incubation. We added 0.1g microplastics into 45ml IS medium,
and added 1ml bacterial solution with OD600 of 0.65 for
co-culture. After co-incubation for 10 days, Fourier Transform
Infrared Spectrometer was used to detect microplastics. It can
be found that the peak of carbon-carbon chain in the
microplastics of the experimental group gradually decreased with
the increase of days, and the oxygen-containing groups gradually
increased, and the change was more obvious than that of the
control group, indicating that Degradation module can play the
role of biodeterioration.

Learn

In this module, we initially wanted to construct a whole plasmid
capable of expressing both AlkB2-Rd45-AdhA and CYPY96F as well
as VHb proteins. To construct the we needed to attach two long
fragments to a vector. At first, we used the enzyme binding
method to make the connection, but we tried for a long time
without success. It was realized that the method of binding two
long f ragments by enzyme digestion was inefficient, and it was
difficult to make the vector express three proteins at the same
time. We changed the way we connected to homologous
recombination and connected one fragment at a time, and we got
the engineering success. The effects of AlkB2-Rd45-AdhA and
CYPY96F were verified respectively.

References

* 1. Teufel, F. et al. SignalP 6.0 predicts all five types of
  signal peptides using protein language models. Nat Biotechnol
  40, 1023-1025 (2022).
* 2. Bergman, M.T., Xiao, X. & Hall, C.K. In Silico Design and
  Analysis of Plastic-Binding Peptides. J Phys Chem B 127,
  8370-8381 (2023).
* 3. Nie, Y., Liang, J., Fang, H., Tang, Y.Q. & Wu, X.L. Two novel
  alkane hydroxylase-rubredoxin fusion genes isolated from a
  Dietzia bacterium and the functions of fused rubredoxin domains
  in long-chain n-alkane degradation. Appl Environ Microbiol 77,
  7279-7288 (2011).

Engineering

Extracellular Electron And CO2 Transfer

{{ generate\_circle\_container(2) }}

Cycle 1:Electron Extracellular Transport

Cycle 2:Carbon Dioxide Transfer

References

Cycle 1: Electron Extracellular Transport

In the anoxic mangrove soil environment,
*Rhodopseudomonas palustris*
CGA009 requires supplementary electron donors for carbon
sequestration alongside carbon dioxide. Consequently, we designed
*P. aeruginosa* PAO1 to act as an electron donor,
facilitating electron transfer to *R. palustris*.

Design

To speed up the carbon sequestration rate of
*R. palustris*, we needed to design a mechanism to allow
electrons from copper-green pseudomonas bacteria to flow to
*R. palustris*. After a basic literature search, we learned
that direct electron transport and indirect electron transport
occurred simultaneously in coculture of *P. aeruginosa* and
*R. palustris*, coupled with each other to jointly increase
the rate of electron transport. Direct electron transfer is
carried out by redox proteins (cytochrome c, biological
nanowires). Indirect electron transfer is through endogenous
electron shuttles (endogenous electron shelters mediated by a
benzoyl substance outside the copper-green *P. aeruginosa*,
and photochrome-mediated endogenous electronic shelterings
outside the *R.palustris* bacteria), exogenous electrons
shuttle and electron carriers. Therefore, we planned to
strengthen both direct and indirect electron transport between
*P. aeruginosa* and *R.palustris*.

![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/1.png)

Figure 1 Gene line diagram of pAB1-pS-*nqrf* plasmid

![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/2.png)

Figure 2 Gene line diagram of pAB1-pS-*pilA* plasmid

Build

The introduction of the nqrf gene into
*P. aeruginosa* induces the production of NADH reductase,
which is also called NADH dehydrogenase, which catalyzes the
transformation of NADH into and electrons, increasing the amount
of electrons in *P. aeruginosa* and thus promoting electron
shuttles (PYO), which gene, was introduced into
*P. aeruginosa*, which can express type 4 whiplash on the
surface of *P. aeruginosa*, form physical contact with
*R. palustris*, conduct direct electron transfer, and
improve the efficiency of electron transfer between
*P. aeruginosa* and *R. palustris*.

![Figure 3  Plasmid construction of pAB-pS-nqrf](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/3.png)

Figure 3 Plasmid construction of pAB-pS-*nqrf*

![Figure 4  Plasmid construction of pAB-pS-pilA](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/4.png)

Figure 4 Plasmid construction of pAB-pS-*pilA*

Test

We used the single fragment homologous recombination method to
assemble the plasmid and obtained the correct colony PCR map
after many attempts.

![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/5.png)
![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/6.png)

Figure 5(left) Electropherogram of pAB1-pS-*nqrf* colony
PCR gel
Figure 6(right) Electropherogram of pAB1-pS-*pilA* colony
PCR gel

Learn

We can find that the *nqrf* gene can increase the content
of NADH reductase in *P. aeruginosa* and accelerate NADH
dehydrogenation to produce and electron content, thereby
increasing the advantage of *P. aeruginosa* as an
electrical organism. After the introduction of the type IV whip
gene , the surface of *P. aeruginosa* can express type IV
whiplash, form physical contact with *R. palustris*,
conduct direct electron transfer, and improve the efficiency of
electron transfer between *P. aeruginosa* and
*R. palustris*.

Cycle 2: Carbon Dioxide Transfer

Design

At room temperature, carbon dioxide exhibits limited solubility
in water, with a ratio of approximately 600:1, primarily forming
carbonic acid and trace amounts of carbonates and bicarbonates.
However, it readily escapes from aqueous solutions when
subjected to heat or agitation, which implies that CO2
emitted by bacteria engineered for mangrove ecosystems can
easily be released into the atmosphere. Furthermore, the
degradation process involving *P. aeruginosa* and
*R. palustris* leads to biofilm formation on microplastics,
significantly impeding CO2 transfer. Consequently, it
is essential to convert CO2 into a form that
facilitates efficient transport within these biofilms.

Build

*Paeruginosa* PAO1 encodes three cytoplasmic
β-carbonic anhydrases (β-CAs):*psCA1*,
*psCA2*, and *psCA3*, which facilitate the reversible
hydration of to bicarbonate, as represented by the equation:
$\small{CO\_2+H\_2O \rightleftharpoons HCO\_3^{-} +
H^+}$.Notably,*psCA1* encoded by the gene *PAO102* or
*psCA1*, is a type I β-CA with superior conversion
efficiency and pH-independent secondary structure. We utilize
*psCA1* to convert , generated by *P. aeruginosa* PAO1
during polyethylene (PE) degradation, into bicarbonate (HCO3-), which is then exported via the BicA transporter.
*R. palustris*, a Gram-negative bacterium with cytoplasmic
and periplasmic compartments, contains an α-CA (encoded by
*acaP*) in its periplasm. This α-CA is inactive under
aerobic conditions but active under anoxic conditions, typical
of the low-oxygen environments where mangrove microplastics
reside. In *R. palustris*, α-CA accelerates the
conversion of HCO3- to , which is
subsequently fixed by ribulose-bisphosphate
carboxylase-oxygenase (RubisCo) into utilizable organic matter.
The ABC transporter substrate-binding protein, a bicarbonate
transporter, is present in the cell membrane of R.palustris,
enabling the uptake of HCO3- and its
conversion back to for the CBB cycle by α-CA. To maximize
processing from microplastic decomposition, we aim to enhance
the HCO3-/CO2.

![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/7.png)

Figure 7 Plasmid construction of pAB-pS-*PAO102*

![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/8.png)

Figure 8 Plasmid construction of pBBR1MCS2-*acap*

Test

We used both single-fragment homologous recombination and
enzymatic ligation methods to assemble the plasmid, and after
several attempts, we obtained the correct colony PCR profiles.

![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/9.png)
![img](https://static.igem.wiki/teams/5291/images/engineering/eet-and-co2/10.png)

Figure 9(left) Electropherogram of pBBR1MCS2-*acaP*
colony PCR gel
Figure 10(right) Electropherogram of
pAB1-pS-*PAO102* colony PCR gel

Learn

Our molecular experiments have been successfully concluded, yet
the forthcoming CO2 absorption studies are
anticipated to demand additional time. As a result, we have not
yet been able to allocate resources to functional validation in
this domain. However, this represents a significant area of
focus for our future enhancements and research endeavors.

References

* 1. Lotlikar, S.R. et al. Three functional β-carbonic anhydrases
  in Pseudomonas aeruginosa PAO1: role in survival in ambient air.
  Microbiology (Reading) 159, 1748-1759 (2013).
* 2. Lotlikar, S.R. et al. Pseudomonas aeruginosa β-carbonic
  anhydrase, psCA1, is required for calcium deposition and
  contributes to virulence. Cell Calcium 84, 102080 (2019).
* 3. Gorby, Y.A. et al. Electrically conductive bacterial
  nanowires produced by Shewanella oneidensis strain MR-1 and
  other microorganisms. Proc Natl Acad Sci U S A 103, 11358-11363
  (2006).
* 4. Pham, T.H. et al. Metabolites produced by Pseudomonas sp.
  enable a Gram-positive bacterium to achieve extracellular
  electron transfer. Appl Microbiol Biotechnol 77, 1119-1129
  (2008).
* 5. Ciemniecki, J.A. & Newman, D.K. NADH dehydrogenases are the
  predominant phenazine reductases in the electron transport chain
  of Pseudomonas aeruginosa. Mol Microbiol 119, 560-573 (2023).

Engineering

Carbon Fixation

{{ generate\_circle\_container(3) }}

Cycle 1:Cellulose Production

Cycle 2:NADPH Biosynthesis

References

Cycle 1: Cellulose Production

Design

Within the framework of microbial carbon sequestration, the
carbon is predominantly sequestered through the formation of
recalcitrant compounds within biological debris. Consequently,
augmenting the biosynthesis and aggregation of cellulose, a
resilient biopolymer, serves to substantially enhance carbon
retention in the soil following the demise of the
microorganisms.

![img](https://static.igem.wiki/teams/5291/images/engineering/carbonfixation/c1.png)

Figure 1 Gene line diagram of pBBR1MCS2-*bcsA-bcsB*
plasmid

Build

Cellulose synthesis usually involves multiple subunits, of which
BcsA and BcsB are the core catalytic subunits responsible for
the synthesis of cellulose chains. Therefore, we increased
cellulose synthesis by transferring to *bcsA* and
*bcsB* derived from Pseudomonas pactida NBRC 14164.

![img](https://static.igem.wiki/teams/5291/images/engineering/carbonfixation/engineering-carbon-fig2.png)

Figure 2 Plasmid construction of pBBR1MCS2-*bcsA-bcsB*

Test

We employed homologous recombination to successfully ligate the
*bcsA* and *bcsB* gene fragments into the vector.
Subsequent verification through colony PCR and sequencing
confirmed the successful ligation of the vector.

![img](https://static.igem.wiki/teams/5291/images/engineering/carbonfixation/c3.png)

Figure 3 Electropherogram of pBBR1MCS2-*bcsA-bcsB* colony
PCR gel

Learn

We initially attempted vector construction through digestion and
ligation, but encountered inefficiencies due to the need for
promoter replacement. Consequently, we redesigned several primer
pairs to facilitate homologous recombination. Despite the
vector-to-fragment ratio being nearly 1:1, which resulted in a
modest recombination efficiency, our persistent refinement of
optimal ligation conditions ultimately enabled us to
successfully construct the desired plasmid.

Cycle 2: NADPH Biosynthesis

Design

The efficiency of cellulose production in *R. palustris* is
limited by the intracellular level of NADPH. However, NADPH
production pathways are limited. It means that cellulose
production is not efficient enough. Therefore, we planned to
increase NADPH biosynthesis in *R. palustris*. Through a
literature search, we chose to introduce the nadK gene from
*R. palustris* RCB100, the pntAB gene from Escherichia
coli, and the nadM gene from Francisella tularensis into
*R. palustris*. By increasing these three genes, NADPH
biosynthesis in engineered bacteria can be enhanced.

![img](https://static.igem.wiki/teams/5291/images/engineering/carbonfixation/c4.png)

Figure 4 Gene line diagram of pBBR1MCS2-*nadK-nadM-pntA-pntB*
plasmid

Build

In *R. palustris*, Nicotinamide-nucleotide
adenylyltransferase (NadM), encoded by the *NadM* gene,
catalyzes the conversion of NMN to NAD+ to prevent
the depletion of pool. *NadK* encoded by nadK
phosphorylates NAD+ to NADP+ and
*pntA-pntB* encoded by PntAB further converts NADP+
to NADPH. It would increase intracellular levels of NADPH in
*R. palustris*. Increased intracellular levels of NADPH
ultimately favor cellulose production.

![img](https://static.igem.wiki/teams/5291/images/engineering/carbonfixation/c5.png)

Figure 5 Plasmid construction of pBBR1MCS2-*nadK-nadM-pntA-pntB*
plasmid

Test

In order to verify the changes in the intracellular level of
NADPH, we divided the *R. palustris* transfected with the
target gene into the experimental group and the ordinary
*R. palustris*
into the control group. We used the NADP+/NADPH
detection kit (NADP+/NADPH Assay Kit with WST-8) to
treated samples and determined changes in the efficiency of
NADPH production by comparing the absorbance of the treated
bacterial samples. Moreover, we also detected the effects of
changes in the intracellular level of NADPH on cellulose
production.

Learn

With these experiments, the data we observed indicates a
significant increase in intracellular levels of NADPH in the
engineered bacteria. It confirms the effectiveness of
*nadK*, *nadM*, and *PntAB*.
Due to the low
success rate of multi-fragment homologous recombination, we
opted to sequentially ligate the inserts, first combining them
and then attaching them to the linearized vector. We have
effectively concatenated the inserts. However, given the
imminent deadline, we faced a time constraint that prevented the
completion of the full experiment. We plan to proceed with the
remaining experimental work following the iGEM competition.

References

* 1. Römling, U. & Galperin, M.Y. Bacterial cellulose
  biosynthesis: diversity of operons, subunits, products, and
  functions. Trends in Microbiology 23, 545-557 (2015).
* 2. Kawai, S., Mori, S., Mukai, T., Hashimoto, W. & Murata, K.
  Molecular characterization of Escherichia coli NAD kinase. Eur J
  Biochem 268, 4359-4365 (2001).

Engineering

Safety

{{ generate\_circle\_container(4) }}

Cycle 1:Primary Designs of the Citrate-activated System

Cycle 2:Optimization of the Promoter of Citrate

Cycle 3:Perfecting of the Suicide System

References

Cycle 1: Primary Design of Citrate-activated System

In order to avoid gene pollution and biological invasion, we
design a safety module to ensure engineered bacteria not to make
environmental pollution.

Design

In order to settle the problem of gene outflow, we conduct some
literature searching and find out several genes for bacteria
apoptosis. At first, we select *bax* as the suicide gene
but the engineered bacteria may be extremely burdened by its
large size. Therefore, we choose out *FADD-DED*, a gene
that is much smaller than *bax* but has the similar effect
with it.

We still take advantage of PcitH to sense the
concentration of citrate and regulate genes downstream.
Moreover, we add some genes like *T7 RNA polymerase* and
*cI* to construct a plasmid anti-loss system, further
preventing gene pollution.

![img](https://static.igem.wiki/teams/5291/images/engineering/the-suicide-system-under-the-normal-condition.png)

Figure 1 The suicide system under the normal condition

![img](https://static.igem.wiki/teams/5291/images/engineering/the-reaction-of-the-suicide-system-when-lacking-in-citrate.png)

Figure 2 The reaction of the suicide system when lacking in
citrate

![img](https://static.igem.wiki/teams/5291/images/engineering/the-plasmid-anti-loss-function-of-the-suicide-system-each-cell-in-the-figure-beyond-represents-a-situation-of-plasmid-loss.png)

Figure 3 The plasmid anti-loss function of the suicide system
(each cell in the figure beyond represents a situation of
plasmid loss)

Build

We divide the gene we interest into two plasmids to lessen the
burden on engineered bacteria.

Viewed as a whole, the promoter PcitH is used to
start the expression of T7 RNA polymerase when there is citrate
existing in peripheral environment, and then T7 RNA polymerase
will stimulate the promoter pT7 so that *cI* can express.
The gene *cI* can produce a kind of substance that
specifically repress the activity of Plam so that it
can inhibit the expression of *FADD-DED* and the bacteria
will survive when citrate is in the environment. If engineered
bacteria are off the citrate system, the decrease of citrate
concentration is not enough to activate PcitH and the
downstream expressions stop. Low expression of *cI* lead to
the re-activation of Plam and *FADD-DED* is able
to come into effect, resulting in cell apoptosis.

Test

To confirm the effect of citrate concentration on
PcitH, we designed a series of citrate concentration
gradient experiments. We link PcitH with GFP and test
the activation of the promoter in different concentration of
citrate through the strength of fluorescent light. However,
things don't turn out as planned due to insensitivity of PcitH
to citrate in *P. aeruginosa*. We speculate that it may
cause by the excessively long sequence we intercept from the
upstream of citH and the promoter cannot come into effect
therefore.

Learn

We plan to find out other citrate-sensitive promoters through
literature searching because of the undesirable sensitivity test
of PcitH in order to realize the regulatory of
citrate concentration to the suicide system.

Cycle 2: Optimisation of the Promoter of Citrate

Due to the weak sensitivity of PcitHto citrate in
*P. aeruginosa*, we have to replace it by another promoter
with similar functions but higher efficiency.

Design

After a long-time literature searching, we select a strong
promoter PopdH from opdH-tctCBA-tctDE manipulator
system, which has the similar ability of citrate-sensitivity
with PcitH but is smaller in volume and more
effective, to replace PcitH. PopdH
natively exists in *P. aeruginosa*, making sure that it can
be activated normally by citrate in the engineered bacteria.
Besides, a new type of bacteria (*R. palustris*) is added
to our design and the opdH-tctCBA-tctDE manipulator system is
absent in it, which means that we need to introduce the
promoter-regulatory gene *tctD* into
*R. palustris* additionally.

Build

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/s4.png)

Figure 4 The new regulatory system in *P. aeruginosa* in
normal conditions

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/s5.png)

Figure 5 The new regulatory system in *R. palustris* in
normal conditions

We follow the previous design of the suicide system but replace
PcitH with PopdH. It is worth mentioning
is that we link the tctD upstream of PopdH in
*R. palustris* so that the promoter is able to be regulated
more accurately.

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/s6.png)

Figure 6 The mechanism of the new system when there is no
citrate (in *P. aeruginosa*)

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/s7.png)

Figure 7 The mechanism of the new system when there is no
citrate (in *R. palustris*)

Test

To confirm the effect of citrate concentration on
PopdH, we designed a series of citrate concentration
gradient experiments. We link PopdH with GFP and test
the activation of PopdH in different concentration of
citrate through the strength of fluorescent light.
Unfortunately, there is a critical deficiency in our design that
*FADD-DED* has a long sequence of C and G base pairs, which
makes it hard to synthase by the method of PCR. Besides, our
design of the plasmid anti-loss system is too large to realize
in labs. For these reasons we have to adjust some genes we apply
and haven't design corresponding test of *FADD-DED* in the
direction of wet lab and dry lab.

Learn

Over these experiments, we learn the importance of simplifying
the system, and we try to design a more straightforward but
still efficient safety module.

Cycle 3: Perfecting of the Suicide System

Owing to the unrealizability of the previous design in labs as
well as the adaptability of the system in engineered
*R. palustris*, we urgently need an easier and more generic
method to realize our wish.

Design

We introduce *hok/sok* toxin-antitoxin system to achieve
both of plasmid anti-loss and bacterial suicide when being out
of the mangrove at the same time. The expression (*sok*
antisense RNA and *hok* mRNA) will specifically bind to
each other and the production of *hok* toxin is inhibited.
However, the half-life of *sok* antisense RNA is much
shorter than that of *hok* mRNA, which results to the
earlier degradation of *sok* antisense RNA than
*hok* mRNA when the plasmid is lost. The *hok* mRNA
remaining is able to encode *hok* toxin and lead to cell
death. We replace the promoter of *sok* with PopdH and the
promoter of *hok* is replaced by a weak promoter PcW common
in P.aeruginosa as well, ensuring the expression strength of
*sok* to be always stronger than the one of *hok* in
normal conditions and cells keeping living. When engineered
bacteria is out of the mangrove, the decrease of citrate
concentration will affect the expression of *sok*, causing
a lower expression level of *sok* antisense RNA. Some of
the *hok*
mRNA is released thus and generates toxin to kill the bacteria.

Build

To secure the plasmid anti-loss effect of the
*hok/sok* system, we attach them to the same plasmid.

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/s8.png)

Figure 8 The new suicide system under normal conditions

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/s9.png)

Figure 9 The effect of the new suicide system when the plasmid
is lost

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/s10.png)

Figure 10 The new suicide system when lacking in citrate

Test

To test the plasmid anti-loss effect of *hok/sok* system,
we design a series of experiments. First, we introduce the
*hok/sok*
system into the experimental group while the empty plasmid
without the system is introduced into the control group, which
enable us to view the killing effect of our design. We take
advantage of the LB liquid culture media to cultivate the both
groups, deliberately making the plasmids in bacteria lost.
Passing the bacteria on until they reach the tenth generation,
we transfer them on the LB solid culture media, cultivating in
37℃ for 16h. Then move the single colonies to those with
ampicillin, cultivating in 37℃ for 6h. Finally, we count the
number of colonies on the plate with ampicillin, and separately
calculate the mortality rate of two groups.

From those test plates, we can make a conclusion that the
mortality rate of the group without *hok/sok* system is
larger than the one with the system. It means that the number of
plasmid-loss bacteria without the system is more than the
experimental group. Therefore, the function of preventing
plasmid loss is effective.

Then we conduct experiments to test the suicide function when
the engineered bacteria are out of the mangrove.

![img](https://static.igem.wiki/teams/5291/images/engineering/safety/fig11ab.png)

Figure 11 (A) The death rate of the groups with and without
hok/sok system, which also means the rate of plasmid loss. (B)
OD600 of the groups with and without IPTG induction, which is
designed as a substitute experiment in the laboratory since
citrate can be consumed by *P. aeruginosa*

Learn

We successfully construct the safety module.

References

* 1. Thisted, T. & Gerdes, K. Mechanism of post-segregational
  killing by the hok/sok system of plasmid R1. Sok antisense RNA
  regulates hok gene expression indirectly through the overlapping
  mok gene. J Mol Biol 223, 41-54 (1992).
* 2. Franch, T., Gultyaev, A.P. & Gerdes, K. Programmed cell death
  by hok/sok of plasmid R1: processing at the hok mRNA 3'-end
  triggers structural rearrangements that allow translation and
  antisense RNA binding. J Mol Biol 273, 38-51 (1997).
* 3. Brocker, M., Schaffer, S., Mack, C. & Bott, M. Citrate
  utilization by Corynebacterium glutamicum is controlled by the
  CitAB two-component system through positive regulation of the
  citrate transport genes citH and tctCBA. J Bacteriol 191,
  3869-3880 (2009).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %} {% block title %}Result{% endblock %} {% block lead
%}You can describe the results of your project and your future plans here.{%
endblock %} {% block page\_content %}

Result

![](https://static.igem.wiki/teams/5291/images/page-header4.svg)
![](https://static.igem.wiki/teams/5291/images/overview/wetlab2.jpg)

Degradation

EET & CO2 Transfer

CO2 Fixation

Safety

Co-culture

Result

Degradation

Plasmid amplification and reconstruction

Strain construction

Expression

Effect verification

Plasmid amplification and reconstruction

In degradation module we constructed 4 main plasmids. Two PE
degrading enzymes, CYPY96F-VHb and AlkB2-Rd45-AdhA, are designed
to accelerate the degradation rate by improving the absorbing and
hydroxylation of PE monomer. In order to express above two enzymes
in our chassis, pAB1-*cypY96F-vgb* and pAB1-*alkB2-Rd45-adhA* were
constructed. pAB1-pS-*PEBP-PEase* was constructed to depolymerize PE
microplastics. In addition, pAB1-*PEBP-GFP* was constructed to
verify the adsorption capacity of PEBP to PE by fluorescence
microscopy.

![img](https://static.igem.wiki/teams/5291/images/result/degradation/a-map-of-plasmid-pab1-alkb2-rd45-adha-b-map-of-plasmid-pab1-cypy96f-vhb-c-map-of-plasmif-pab1-ps-pebp-gfp-d-map-of-pab1-ps-pebp-pease.png
                )

Figure 1 a. Map of plasmid pAB1-*alkB2-Rd45-adhA* b. Map of
plasmid pAB1-*cypY96F-vgb* c. Map of plasmif pAB1-pS-*PEBP-GFP* d.
Map of pAB1-pS-*PEBP-PEase*

![img](https://static.igem.wiki/teams/5291/images/result/degradation/the-pcr-result-of-alkb-rd45-adha-fragment-the-band-was-identycal-to-the-expected-length-of-3033bp.png)

Figure 2 The PCR result of AlkB-Rd45-AdhA fragment. The band was
identical to the expected length of 3033bp

![img](https://static.igem.wiki/teams/5291/images/result/new/f3.png)

Figure 3 The PCR result of CYPY96F-VHb fragment. The band was
identical to the expected length of 2285bp

![img](https://static.igem.wiki/teams/5291/images/result/degradation/the-pcr-result-of-pebp-gfp-fragment-the-band-was-identyca-to-the-expected-length-of-2108bp.png
                )

Figure 4 The PCR result of PEBP-GFP fragment. The band was
identical to the expected length of 2108bp

![img](https://static.igem.wiki/teams/5291/images/result/degradation/the-pcr-result-of-pebp-gfp-fragment-the-band-was-identyca-to-the-expected-length-of-3231bp.png
                )

Figure 5 The PCR result of PEBP-GFP fragment. The band was
identical to the expected length of 3231bp

The bands of AlkB2-Rd45-AdhA, CYP-VHb, PEBP-GFP and PEBP-PEase,
from PCR were identical to the theoretical lengths estimated by
the designed primer locations, which could demonstrate that we
successfully amplified our target fragments, we then used
homologous recombination to link the vectors and fragments.

Strain construction

We transferred the constructed plasmids into
*Escherichia coli*
DH5αstrain and conducted colony PCR. After obtained the
correct results we amplified and extracted the constructed
plasmids. Then we transferred these plasmids into
*Pseudomonas aeruginosa* PAO1 strain and *E. coli* BL21
strain and obtained correct colony PCR results, indicating that we
successfully constructed strains containing these plasmids.

![img](https://static.igem.wiki/teams/5291/images/result/new/f6.png)

Figure 6 *P.aeruginosa* PAO1 colony PCR results of
CYPY96F-VHb and AlkB2-Rd45-AdhA

![img](https://static.igem.wiki/teams/5291/images/result/new/f7.png)

Figure 7 *P.aeruginosa* PAO1 colony PCR result of
PEBP-PEase and PEBP-GFP. In order to improve primer specificity,
we change the location of primer and PEBP-GFP theoretical
lengths estimated by the designed primer locations is 1875bp

Expression

In order to verify whether AlkB2-Rd45-AdhA, CYPY96F and VHb
proteins were successfully expressed in the strains, SDS-PAGE was
performed and the following results were obtained.

![img](https://static.igem.wiki/teams/5291/images/result/new/f8.png)

Figure 8 SDS-PAGE result of CYPY96F-VHb

The plasmid pAB1-*cypY96F-vgb* is expected to express CYPY96F and
VHb. The molecular weight of CYPY96F is estimated to be 47.48kDa
and that of VHb is estimated to be 16.73kDa. In the figure, it can
be seen that the bands in the pAB1-*cypY96F-vgb* group were
significantly deepened at around 45kDa and 15kDa compared with the
control group, indicating that CYPY96F and VHb proteins were
successfully expressed.

![img](https://static.igem.wiki/teams/5291/images/result/degradation/sds-page-result-of-alkb2-rd45-adha.png)

Figure 9 SDS-PAGE result of AlkB2-Rd45-AdhA

Plasmid pAB1-AlkB2-Rd45-AdhA is expected to express a large fusion
protein AlkB2-Rd45-AdhA with a molecular weight of 94.59kDa. In
the figure, it can be seen that there were an extra bands in the
experimental group than in the control group at a position
slightly less than 100kDa, suggesting that AlkB2-Rd45-AdhA protein
was successfully expressed.

![img](https://static.igem.wiki/teams/5291/images/result/degradation/sds-page-result-of-pebp-gfp.png)

Figure 10 SDS-PAGE result of PEBP-GFP

The plasmid pAB1-pS-*PEBP-GFP* is expected to express a large fusion
protein PEBP-GFP protein with a molecular weight of 67.73kDa. In
the figure, it can be seen that compared with the control group,
the experimental group had multiple marker bands in the middle of
the marker bands slightly smaller than 75kDa and 60kDa, suggesting
that the PEBP-GFP protein was successfully expressed.

The plasmid PAB1-pS-*PEBP-PEase* was expected to express a protein
with a size of 188.18kDa. Unfortunately, we did not see the
corresponding bands, so the protein may have failed to be
expressed in bacteria. We hypothesized that the protein could not
be expressed correctly due to its large molecular weight.
Therefore, we will verify the related effects of PEBP mainly by
engineering bacteria with plasmid pAB1-pS-*PEBP-GFP*.For the part of
the PEBP-PEase protein that are not verified, we used modeling
methods to predict it.

Therefore, in the expression verification stage, we successfully
verified the expression of AlkB2-Rd45-AdhA, CYPY96F, VHb and
PEBP-GFP proteins, which laid a solid foundation for our
subsequent effect verification.

Effect verification

In order to verify that the engineering bacteria did degrade the
microplastics faster, a 7-day co-incubation experiment was
conducted. When the co-culture was done, microplastics were
carefully separated from the culture medium and weighed. Fourier
infrared (FTIR) detection was performed on the incubated
microplastics to check the property changes of the microplastics,
and scanning electron microscopy was used to detect the
morphologic change on the surface of the microplastics.

Weighing:

The results of the weighing were only slightly different. The
remaining microplastics separated weighted 0.094g, 0.094g and
0.098g in PAO1*::alkB2-Rd45-adhA*,
*PAO1::cypY96F-vgb,* wild type PAO1 group. This result may
not be very convincing but it may also hint that our engineered
bacteria can degrade microplastics more effectively.

Fourier infrared detection:

![img](https://static.igem.wiki/teams/5291/images/result/degradation/ftir-results-of-co-cultured-pe-microplastics.png)

Figure 11 FTIR results of co-cultured PE microplastics \*The peak
around 2400 cm-1 is caused by the instrument itself,
that it lacks of argon

The FTIR results show that, the group without any bacteria have
shown classic absorption peaks of PE. The characteristic PE
absorbance peaks are located at 2921 cm-1, 2851
cm-1, 1456 cm-1, and 722cm-1,
each standing for CH2 asymmetric C-H stretch, CH2
symmetric C-H stretch, CH3 umbrella bending mode, and
spilt CH2
rock.

Compared to its line, the bacteria input group's peaks have
decreased in the intensity level, indicating the decrease in
chemical bonds. Especially, the engineered PAO1 have shown lower
intensity than the wild type of PAO1. The decrease of chemical
bonds indicates that the PAO1 is able to degradant PE, and our
plasmids have improved its ability.

Scanning electron microscopy:

![img](https://static.igem.wiki/teams/5291/images/result/degradation/from-left-to-right.png)

Figure 12 From left to right, PAO1*::alkB2-Rd45-adhA*,
PAO1*::cypY96F-vgb*, wild type PAO1, blank control,
respectively In the first row, the experimental group is magnified
900 times, the control group is magnified 300 times In the second
row, the experimental group was magnified 5,000 times and the
control group 2,500 times

It can be seen that the overall particle size of microplastics
degraded by engineering bacteria is generally smaller than that of
the control group and microplastics co-incubated with Engineering
bacteria were broken into small fragments while control group
maintains complete microplastic particles. In the enlarged images
the microplastics co-incubated with the engineering bacteria
displayed a rough surface, while the microplastic in the control
group is rather smooth. All of these features suggest that
engineering bacteria degrade plastics more efficiently.

Effect verification of PEBP:

![img](https://static.igem.wiki/teams/5291/images/result/degradation/from-left-to-right-are-images-of-microplastics-with-500mm-diameter-incubated-with-gfp-with-pe-binding-peptide-with-gfp-protein-alone-and-a-blank-control.png)

Figure 13 From left to right are images of microplastics with
500mm diameter incubated with GFP with PE binding peptide, with
GFP protein alone, and a blank control

The PEBP-GFP protein was extracted and purified. After being
immersed in PBS and cleaned twice by the vortex shaker, the
microplastics incubated with PEBP-GFP still generally showed
obvious fluorescence. The microplastics incubated with GFP can see
weak fluorescence of GFP residue after intense exposure, while the
blank group can not see any fluorescence under the fluorescence
microscope. It can be concluded that PEBP has strong binding
ability with microplastics.

Modeling of PEBP-PEase:

![img](https://static.igem.wiki/teams/5291/images/result/degradation/1536-1727270912.gif)

Figure 14 Predicted structure of PEBP-PEase protein

For PEase, we tested its effect through moduling. We conducted
structure predicting of PEBP-PEase. The predicting module clearly
displayed a membrane spanning domain and an enzyme domain, which
we exactly expected our bacteria to produce. After that molecular
docking was conducted to study the interactions between the PEase
enzyme and both large and small alkane molecules. It can be
observed that the docking sites and the binding pockets are
generally located within the same binding pocket, with multiple
amino acid residues (such as LEU246, ILE250, VAL589, LEU622, etc.)
surrounding the long-chain alkane. These amino acids are mostly
hydrophobic residues, which likely stabilize the alkane molecules
through hydrophobic interactions. Their affinity suggests that
PEase may have the ability to bind to and degrade PE
microplastics. (For detail information, please visit Modeling
page)

Result

Extracellular Electron And CO2 Transfer

Plasmid amplification and reconstruction

Strain construction

Expression

Validation

1.Calculation of NAD + / NADH amount

2. Flagella silver staining

3. MFC Testing

We engineered plasmids pAB1-pS-*pilA* and pAB1-pS-*nqrf*
to boost electron transfer in *P. aeruginosa* and
*R. palustris*, featuring a truncated *pilA* gene with
mutations (E32Y, L51F, G57Y) and increased *nqrf* expression.
Additionally, we developed plasmids pAB1-pS-*PAO102* and
pAB1-pS-*acap* to enhance CO2 transfer in
*P. aeruginosa* and uptake in *R. palustris*.

Plasmid amplification and reconstruction

The introduction of the pAB1-pS-*nqrf* plasmid into
*P. aeruginosa* induces the production of NADH dehydrogenase,
which catalyzes the transformation of NADH into and electrons,
increasing the amount of electrons in *P. aeruginosa* and
thus promoting electron shuttles (PYO), which indirectly transmit
electrons to *R. palustris*.

![img](https://static.igem.wiki/teams/5291/images/result/plasmid-construction-of-pab1-ps-inqrfi.png)

Figure 1 Plasmid construction of pAB1-pS-*nqrf*

![img](https://static.igem.wiki/teams/5291/images/result/plasmid-construction-of-pab1-ps-pila.png)

Figure 2 Plasmid construction of pAB1-pS-*pilA*

![img](https://static.igem.wiki/teams/5291/images/result/plasmid-construction-of-pab1-ps-pao102.png)

Figure 3 Plasmid construction of pAB1-pS-*PAO102*

![img](https://static.igem.wiki/teams/5291/images/result/plasmid-construction-of-pab1-ps-acap.png)

Figure 4 Plasmid construction of pAB1-pS-*acap*

Strain construction

| Plasmids | Expected size(bp) | Fragments | Expected size(bp) |
| --- | --- | --- | --- |
| pAB1-pS-*nqrf* | 9071 | *nqrf* | 1245 |
| pAB1-pS-*pilA* | 7810 | *pilA* | 201 |
| pAB1-pS-*PAO102* | 8381 | *PAO102* | 747 |
| pAB1-pS-*acap* | 5944 | *acap* | 837 |

Table 1 Plasmids constructed in the extracellular electron and
carbon dioxide transfer module

We successfully constructed all the plasmids mentioned in the
table and below are their colony PCR electrophoresis and gene line
diagrams.

![img](https://static.igem.wiki/teams/5291/images/result/agarose-gel-electrophoresis-analysis-of-pab1-ps-nqrf.png)

Figure 5 Agarose gel electrophoresis analysis of pAB1-pS-*nqrf*

![img](https://static.igem.wiki/teams/5291/images/result/agarose-gel-electrophoresis-analysis-of-pab1-ps-pila.png)

Figure 6 Agarose gel electrophoresis analysis of pAB1-pS-*pilA*

![img](https://static.igem.wiki/teams/5291/images/result/agarose-gel-electrophoresis-analysis-of-pab1-ps-pao102.png)

Figure 7 Agarose gel electrophoresis analysis of pAB1-pS-*PAO102*

![img](https://static.igem.wiki/teams/5291/images/result/agarose-gel-electrophoresis-analysis-of-pab1-ps-acap.png)

Figure 8 Agarose gel electrophoresis analysis of pAB1-pS-*acap*

Expression

SDS-PAGE analysis of the induced cells after the construction of
the expression vectors showed that the Nqr(46.40 kDa) and
*pilA*(12.08 kDa) proteins had the expected molecular
weights.

![img](https://static.igem.wiki/teams/5291/images/result/sds-page-analysis-of-the-expression-of-pab1-ps-nqrf-pab1-ps-pila-in-pao1.png)

Figure 9 SDS-PAGE analysis of the expression of
pAB1-pS-*nqrf*,pAB1-pS-*pilA* in PAO1

Validation

1.Calculation of NAD+ / NADH amount

We used NADH Assay Kit with WST-8 to measure the amount of
NAD+ / NADH in *P. aeruginosa* and
*P. aeruginosa*/pAB-pS-*nqrf*. By comparing the
proportion of NADH in the control group and the experimental
group, we can find that NADH/(NADH + NAD+ ) in
experimental group is lessen than control group, which means
*P. aeruginosa* with pAB-pS-*nqrf* plasmid can product
more NADH reductase and electronics. As shown in the Figure 10
and Table 2.

![img](https://static.igem.wiki/teams/5291/images/result/nadh-nadh-nad-amount-of-pao1pab-ps-nqrf-pao1.png)

Figure 10 NADH, NADH+NAD+amount of
PAO1/pAB-pS-*nqrf*, PAO1

| Concentration(μM)  Group | Experimental Group | Control Group |
| --- | --- | --- |
| NADH + NAD+ | 0.217 | 0.116 |
| NADH | 0.131 | 0.0917 |
| NADH/(NADH + NAD+) | 0.604 | 0.793 |

Table 2 NADH + NAD+ amount, NADH amount, NADH/(NADH
+ NAD+) of PAO1/pAB-PS-*nqrf*, PAO1

2. Flagella silver staining

We stained the flagella using silver staining, which was used to
verify the validity of our plasmid pAB-pS-*pilA*. After
staining, we found that the flagellum length of the experimental
group was longer and larger than that of the control group,
which was in line with our experimental expectations.

![img](https://static.igem.wiki/teams/5291/images/result/light-microscopy-to-visualize-the-bacterial-surface.png)

Figure 11 Light microscopy to visualize the bacterial surface
flagella PAO1 and
PAO1/pAB1-pS-*pilA*,magnification:40$\times$10.

3.MFC Testing

We measured the conductivity of the bacterial fluid by measuring
the MFC experiment and found that the voltage of the bacterial
fluid with the introduction of plasmid *pilA* was
significantly higher than the voltage of the original bacteria
over time. This proves that our plasmid is effective in
enhancing the electron conduction function of
*P. aeruginosa*, and this experiment met our expectations.

Therefore, compared to *P. aeruginosa* that did not import
plasmids, plasmid-infiltrated *P. aeruginosa* had more
advantages as electrical donator and were more conducive to
extracellular transfer between *R. palustris*.

![img](https://static.igem.wiki/teams/5291/images/result/the-out-put-voltage-of-pao1-pao1.png)

Figure 12 The out put voltage of
PAO1,PAO1/*nqrf*+,PAO1/*pilA*+

Result

Carbon dioxide fixation

Plasmid amplification and reconstruction

Strain construction

1.pBBR1MCS2-*bcsA*-*bcsB*

2.pBBR1MCS2-*nadM*-*nadK*-*pntA*-*pntB*

Expression

Validation

1. Cellulose FT-IR Analysis

2. Congo red staining

Plasmid amplification and reconstruction

| Plasmids | Expected size(bp) | Fragments | Expected size(bp) |
| --- | --- | --- | --- |
| pBBR1MCS2-*bcsA*-*bcsB* | 10024 | *bcsA*-*bcsB* | 4892 |
| pBBR1MCS2-*nadM*-*nadK*-*pntA*-*pntB* | 7810 | *nadM* | 1044 |
| *nadK* | 825 |
| *pntA*-*pntB* | 2932 |

Table 1 Plasmids constructed in the Carbon Dioxide Fixation
module

1.We constructed the plasmid pBBR1MCS2-*bcsA*-*bcsB* for
cellulose production with the goal of carbon dioxide fixation.

![img](https://static.igem.wiki/teams/5291/images/result/schematic-diagram-of-the-pbbr1mcs2.png)

Figure 1 Schematic diagram of the pBBR1MCS2-*bcsA*-*bcsB*
plasmid

2.To increase the intracellular level of NADPH, we constructed a
plasmid containing four exogenous genes, including *nadM*,
*nadK*, *pntA*, and *pntB*. Here we successfully
amplified the target gene.

![img](https://static.igem.wiki/teams/5291/images/result/pbbr1mcs2-plasmid.png)

Figure 2 Schematic diagram of the
pBBR1MCS2-*nadM*-*nadK*-*pntA*-*pntB*
plasmid

Strain construction

1.pBBR1MCS2-*bcsA*-*bcsB*

![img](https://static.igem.wiki/teams/5291/images/result/agarose-gel-electrophoresis-analysis-of-pbbr1mcs2.png)

Figure 3 Agarose gel electrophoresis analysis of
pBBR1MCS2-*bcsA*-*bcsB*(3226bp)

Due to the challenges associated with primer placement at the
extremities of the target sequence, we designed primers for
colony PCR to amplify a **3,226 base pair (bp) fragment**.
The resulting agarose gel electrophoresis pattern confirmed the
accuracy of the colony PCR, indicating successful amplification
of the intended DNA sequence.

2.pBBR1MCS2-*nadM*-*nadK*-*pntA*-*pntB*

![img](https://static.igem.wiki/teams/5291/images/result/gene-line-diagram-of-pbbr1mcs2.png)

Figure 4 Gene line diagram of
pBBR1MCS2-*nadM*-nadN-*pntA*-*pntB*

![Image 1](https://static.igem.wiki/teams/5291/images/result/agarose-gel-electrophoresis-of-digested-fragments-of-pbbr1mcs2.png)

Figure 5 Agarose gel electrophoresis of digested fragments
of pBBR1MCS2

![Image 2](https://static.igem.wiki/teams/5291/images/result/electropherogram-of-nadm-nadk-pcr-gel.png)

Figure 6 Electropherogram of *nadM*-*nadK* PCR
gel

![Image 3](https://static.igem.wiki/teams/5291/images/result/electropherogram-of-pnta-pntb-pcr-gel.png)

Figure 7 Electropherogram of *pntA*-*pntB* PCR
gel

![Image 4](https://static.igem.wiki/teams/5291/images/result/electropherogram-of-nadm-nadk-pnta-pntb-pcr-gel.png)

Figure 8 Electropherogram of
*nadM*-*nadK*-*pntA*-*pntB* PCR gel

Since the efficiency of homologous recombination of multiple
fragments was too low, we decided to ligate the two inserts
first, and then ligate the inserts to the linearized vector.Here
we have successfully concatenated the inserts.

As the deadline was already approaching, the time required to
complete the experiment was insufficient. We will continue the
subsequent experiments after the iGEM competition.

Expression

SDS-PAGE analysis of the induced cells after the construction of
the expression vectors showed that the *bcsA*,*bcsB*
proteins had the expected molecular weights.

![img](https://static.igem.wiki/teams/5291/images/result/sds-page-analysis-of-the-expression-of.png)

Figure 9 SDS-PAGE analysis of the expression of *bcsA* (83
kd) and *bcsB* (95 kd) in *E.coli* BL21

Validation

1. Cellulose FT-IR Analysis

We used engineered and unengineered bacteria as experimental and
control groups for a three-day cellulose production culture.
After three days, we filtered, separated and dried the cellulose
contained in 30 ml of the bacterial solution for FT-IR
analysis.The characteristic peaks of the infrared Fourier
spectrum (FTIR) of cellulose are mainly distributed in the range
of 3300-3500 cm-¹ (-OH stretching vibration), 2900 cm-¹ (methyl
or methylene stretching vibration), 1640-1650 cm-¹ (water
molecule bending vibration), 1500-1600 cm-¹ (C-O stretching
vibration), 1375-1425 cm-¹ (methylene bending vibration)
1500-1600 cm-¹ (C-O stretching vibration), 1375-1425 cm-¹
(methylene bending vibration), and the regions of 1160-1200 cm-¹
and 1100-1030 cm-¹ (C-O-C skeleton vibrations) and other
regions.The characteristic peaks were evident in the
experimental group, whereas in the control group, due to the
problem of very small yields, the characteristic peaks were
measured to be unclear.

![img](https://static.igem.wiki/teams/5291/images/result/comparison-of-cellulose-ftir-analysis-between-experimental-and-control-groups.png)

Figure 10 Comparison of cellulose FTIR analysis between
experimental and control groups

2. Congo red staining

When cellulose is present, Congo red forms a red complex with
it, causing the medium to appear red.Since we have not been able
to resuscitate *R. palustris* very well, we have temporarily
tested it in *E. coli*.

![img](https://static.igem.wiki/teams/5291/images/result/congo-red-staining-of-dh5-pbbr1mcs2.png)

Figure 11 Congo red staining of
DH5α/pBBR1MCS2-*bcsA*-*bcsB*,DH5α,DH5α/pBBR1MCS2.

The engineered bacteria
DH5α/pBBR1MCS2-*bcsA*-*bcsB*, as well as the
individual *E. coli* DH5α and
*E. coli* DH5α transformed with the empty vector
pBBR1MCS2, were subjected to Congo red staining. The results
showed that the engineering bacteria produced obvious red areas
after staining, while *E. coli* DH5α alone did not
show red, and there was a non-obvious red halo around the outer
ring of *E. coli* transferred to the empty vector, thereby
confirming the cellulose production capability of the engineered
bacteria.

Result

Safety

Plasmid amplification and reconstruction

Strain construction

Validation

Plasmid amplification and reconstruction

We have constructed two plasmids. One is pAB1-PopdH,
which is aiming to validate the effect of citrate sensation of the
promoter PopdH; the other is
pAB1-*hok*/*sok*, which is used to limit the activity
range of engineered bacteria as well as the realization of plasmid
anti-loss. And we obtain the corresponding vector fragments by the
digestion of restriction endonuclease, which are separately used
as the backbone of the vectors for reverse. We get the DNA
sequences we need (*hok*/*sok* and PopdH,
both of them have homologous arms) through PCR.

![img](https://static.igem.wiki/teams/5291/images/result/first.png)

Figure 1 Plasmid construction of pAB1-popDH-hok/sok

Strain construction

After having access to the elements that we use to construct the
plasmids, we conduct the homologous recombination to form complete
plasmids as well as plasmid transfection. Then we conduct colony
PCR. The schemas of the DNA sequences and the results of colony
PCR are attached below.

![img](https://static.igem.wiki/teams/5291/images/result/fig1.png)

Figure 2 The schema of *hok*/*sok* and the result of
corresponding colony PCR.

![img](https://static.igem.wiki/teams/5291/images/result/fig2.png)

Figure 3 The schema of PopdH and the result of
corresponding colony PCR.

Validation

**1. the system of plasmid anti-loss**

We conduct the verification experiment of the plasmid anti-loss
function of *hok*/*sok* system. We draw some squares on
the LB-Amp plates and inoculate the bacteria with and without
*hok*/*sok* system into the squares to examine whether
the plasmid with Amp resistant gene still exists in cells or not.

![img](https://static.igem.wiki/teams/5291/images/result/ab.png)

Figure 4 (A) The photo of the plate of 17th
generation. (B) The result of the plasmid anti-loss experiment

**2. the sensation of PopdH to citrate**

To verify the citrate-sensation function of PopdH, we
conduct bacteria-citrate coincubation, observing the fluorescence
intensity in different gradients of concentration. Taking
advantage of enzyme labeling apparatus, we examine the difference
in fluorescence intensity of each group and create the diagram
below.

We can easily know from the fig.6(B) that the death rate of
bacteria with *hok*/*sok* system is lower than the
control group with the increasing generation, which means that the
*hok*/*sok* system we design can exactly prevent plasmid
loss.

![img](https://static.igem.wiki/teams/5291/images/result/popdh-fluorescence-intensity.png)

Figure 5 The fluorescence intensity variability analysis chart

From the chart we can know from the result that the group without
citrate activation has essentially no fluorescence compared to
pure broth. However, the groups with citrate activation fluoresce
to varying degrees, in which the group of 5mM citrate
concentration has the strongest fluorescence. We can make a
conclusion that citrate is actually able to activate the function
of PopdH, and we achieve our goal of regulating genes
based on the concentration of citrate in the environment.

**the system of suicide when there is no citrate (or activators)
in the environment**

Given that *Pseudomonas aeruginosa* is able to take use of
citrate as a kind of carbon source, we introduce *pLac* in
front of *sok* for research convenience. We separately
cultivate engineered bacteria with the plasmid pAB-*hok*/*sok*
in LB-Amp broth with and without IPTG in the broth and examine
OD600 of both groups every 16h using the enzyme labeling
apparatus. The corresponding diagram is attached below. The
concentration of IPTG we use is 0.6mM.

![img](https://static.igem.wiki/teams/5291/images/result/fig3.png)
![img](https://static.igem.wiki/teams/5291/images/result/hoksok-differences.png)

Figure 6 The different OD600 of groups with and without IPTG

We can know from the diagram that the OD600 of the group with IPTG
is much higher than the other group, which means that IPTG is able
to induce the expression of *sok* and secure the living of
bacteria. Therefore, the system of conditional suicide is
effective.

Result

Co-culture

Co-culture experiments with *P. aeruginosa* and
*R. palustris*

Co-culture power generation experiments

Co-culture experiments with *P. aeruginosa* and
*R. palustris*

The ultimate requirement for the system we constructed is that the
two bacteria are able to work together, so it is important to
explore the optimal co-culture conditions. We could not find any
examples of co-culture of related bacteria, so we set up our own
co-culture medium with a volume ratio of LB medium: ATYP medium of
1:1 for the growth of the two bacteria and performed a growth
curve test for 72 hours. In addition to the choice of medium, we
also considered other factors such as temperature, whether to
oscillate the culture or not, whether to light or not, and finally
chose the culture conditions of 27 degrees Celsius, light,
anaerobic for the *R. palustris* and aerobic for the
*P. aeruginosa* in the static culture program.

The final growth curve results show that both bacteria can grow
effectively in the co-culture medium, and the growth rate of
*P. aeruginosa* is faster than that of *R. palustris*,
which is in line with the reality, so it can be proved that the
results of the co-culture experiment have reached the experimental
expectations, which lays the foundation for our work of testing
the voltage later.

![img](https://static.igem.wiki/teams/5291/images/result/co-culture/co-culture-growth-curves-for-72-hours.jpg)

Figure 1 Co-culture growth curves for 72 hours

Co-culture power generation experiments

In order to test whether the extracellular electron transfer
between the two bacteria can be efficiently carried out, we
prepared engineered *P. aeruginosa* possessing genes that
promote electron transfer for voltage determination. For the first
short period of time at the beginning of the experiment, we added
only the *P. aeruginosa*, and when the voltage was
stabilized, we added the *R. palustris* in an attempt to
observe the changes produced by this manipulation of the voltage.
The experimental data revealed that the engineered
*P. aeruginosa* strain harboring the nqrf gene initially
generated a substantial voltage, indicative of robust metabolic
activity. However, upon the introduction of *R. palustris*,
there was a notable decrease in voltage by approximately 5000 mV,
followed by a fluctuating voltage distribution that stabilized
around a constant mean value. This observation suggests that the
voltage shift post-addition of *R. palustris* might be
indicative of inter-bacterial electron transfer, leading to a
diminished multimeter response.

In a parallel experiment, the *R. palustris* strain
engineered to contain the pilA gene exhibited stronger voltages
compared to the control. Nevertheless, no significant alteration
in solution conductivity was detected after the addition of the
*R. palustris* culture. We speculate that the primary reason
for this discrepancy is the prolonged soaking of the
cation-exchange membrane, which may have compromised its
integrity. However, due to the limited availability and the high
value of the experimental material, coupled with time constraints,
we have not been able to replicate this series of experiments. We
intend to address this in future studies to further elucidate the
observed phenomena.

![img](https://static.igem.wiki/teams/5291/images/result/co-culture/output-voltage-of-co-culture-ir-palustrisi-solution.png)

Figure 2 Output voltage of co-culture
*R. palustris* solution

{% endblock %}