2022/7/4
Today we received the plasmid pGEX-4T-1 from vendor. We quantified the concentration of the plasmid for further use. (AH, FG, QG, WC)

2022/7/5
Today we transformed Escherichia coli K-12 TOP10 using plasmid pGEX-4T-1. The bacteria were cultured at 37℃ overnight. (AH, FG, QG, WC)

2022/7/6
Today we extracted the plasmids inside the transformed bacteria. The extracted plasmids were digested by restriction endonuclease II. Gel electrophoresis was also conducted to test the reliability of previous transformation.

The result was not quite satisfying due to misoperation. (AH, WC)
The experiments conducted these days help us get familiar with some basic biological techniques.

2022/7/8
Preparing LB agar plates with chloramphenicol. (AH)

2022/7/9
Culture TOP10 cells with pGEX-4T-1 (AH, WC)

2022/7/11
Transforming pCas9 into E. coli MG1655. (FG, QG)

2022/7/14
Preparing LB broth, LB broth with chloramphenicol. (AH)
We started to culture E. coli MG1655 pGEX-4T-1-CsgA-MBP3 to test biofilm formation. (AH, WC)

2022/7/15
Obtain LB broth with ampicillin. (AH)
Culturing E. coli MG1655 pCas9. Using glycerol and LB broth to preserve E. coli MG1655 pCas9. (FG, QG)

2022/7/16
Conducting colony polymerase chain reaction (PCR) and agarose gel electrophoresis on E. coli MG1655 pGEX-4T-1. (AH, WC)

2022/7/18
Testing biofilm formation of E. coli MG1655 pGEX-4T-1-CsgA-MBP3. (AH, WC)

2022/7/19-20
Extracting RNA from E. coli MG1655 pGEX-4T-1-CsgA-MBP3. Conducting reverse transcription and real-time quantitative PCR (RT-qPCR) (AH, WC)

2022/7/22
Obtain TE buffer. (AH)

2022/7/26
Conducting colony PCR on E. coli MG1655 pGEX-4T-1-CsgA-MBP3. Result not satisfying. (AH)

2022/7/27
Incubating E. coli MG1655 pGEX-4T-1-CsgA-MBP3. Inoculating on LB agar plates. (AH, WC)

2022/7/28
Conducting colony PCR on E. coli MG1655 pGEX-4T-1-CsgA-MBP3 cultured at 2022/7/27. (Ah, WC)

2022/7/29-8/7
Biofilm growth quantification using dry weight variation method. (AH, WC)

2022/8/2-3
Conducting scanning electron microscopy (SEM) on E. coli MG1655 pGEX-4T-1-CsgA-MBP3 to check whether it had bound metal nanoparticles (NPs). (WC)

2022/8/5
Obtain silver nitrate stock solution. (AH)

2022/8/8
Obtain LB broth with ampicillin. (AH)

2022/8/9
Transformation pCRISPR into E. coli MG1655 pCas9. (FG, QG)

2022/8/9-12
Staining the biofilm using crystal violet. Using microplate reader to test absorbance. (AH, WC)

2022/8/10
Transforming E. coli MG1655 using pGEX-4T-1-CsgA-MBP5. (AH, WC)

2022/8/13
Culturing E. coli MG1655 pGEX-4T-1-CsgA-MBP5. (AH, WC) Extracting RNA from E. coli MG1655 pCas9-pCRISPR. (FG, QG)

2022/8/14-15
Conducting colony PCR on E. coli MG1655 pGEX-4T-1-CsgA-MBP5. (AH)

2022/8/15
Conducting qPCR on E. coli MG1655 pCas9-pCRISPR to check whether the knockout of gene CsgA is successful or not. (FG, QG)
It succeeded.

2022/8/16
Using UV-vis to test the existence of AgNPs. (WC)
Culture E. coli MG1655 pGEX-4T-1-CsgA-MBP3 with Ag⁺ and E. coli MG1655 pGEX-4T-1-CsgA-MBP3 with [AuCl₄]^-. (AH, WC)

2022/8/18
Extracting RNA from E. coli MG1655 ΔCsgA. (WC)

2022/8/21
Conducting qPCR on E. coli MG1655 ΔCsgA to check the knockout result. (FG, QG)

2022/8/22
Culturing E. coli MG1655 pGEX-4T-1-CsgA-MBP3 with Ag⁺ and E. coli MG1655 pGEX-4T-1-CsgA-MBP3 with [AuCl₄]^- using a different metal concentration with 2022/8/16. (AH, WC)

2022/8/24
Transforming pET28a-SUMO-shMt into E. coli MG1655 ΔCsgA. Obtain metal ions stock solution. (FG, QG)

2022/8/25
Conducting inductively coupled plasma mass spectrometry (ICP-MS) on 2022/8/16 samples. (AH, WC) Transforming pET28a-SUMO-shMt and pET28a-SUMO-SmtA into E. coli MG1655 and E. coli MG1655 ΔCsgA. (FG, QG)

2022/8/26
Conducting ICP-MS on 2022/8/22 samples. (AH, WC)

2022/8/27 Culturing E. coli MG1655 pGEX-4T-1-CsgA-MBP3 with Ag⁺ and E. coli MG1655 pGEX-4T-1-CsgA-MBP3 with [AuCl₄]^- Using the same metal concentration as 2022/8/22. (AH)

Transforming: pET28a-SUMO-SmtA, pET28a-SmtA, pET28a-shMt into E. coli MG1655 ΔCsgA. pET28a-SmtA and pET28a-shMt into E. coli MG1655. Conducting colony PCR to verify pET28a-SUMO-shMt inside E. coli MG1655 ΔCsgA. (FG, QG)

2022/8/28
Culturing bacteria under different concentration of metal ions. (FG, QG)

2022/8/29
Conducting colony PCR on pET28a-SUMO-SmtA in E. coli MG1655 ΔCsgA. (FG, QG)

2022/8/30
Using ICP-MS to test the concentration of metal ions in supernatant of culture at 2022/8/28. (FG, QG)

2022/9/1
Same as 2022/8/27. (AH, WC)

2022/9/3
Measure the growth curve of 5 different bacteria in presence of copper ion (II). (FG, QC)

2022/9/3-4
Using microplate reader to test the growth of E. coli together with silver ion (I) and complexed gold ion (III). (AH, WC)

2022/9/4
Conducting qPCR to verify expression of SmtA, SUMO-SmtA and shMt. (FG, QG)

2022/9/5
Microwave digestion of cultures from 2022/9/3. (AH, FG, QG)

2022/9/6
Conducting ICP-MS on samples obtained at 2022/9/5. (FG, QG)

2022/9/7
Culturing E. coli shMt, SUMO-shMt, SUMO-SmtA and SmtA with heavy metals (Cu(II), Cd(II)). (FG, QG)

2022/9/9
Assessment of E. coli metal tolerance. Conducting RT-qPCR for pET28a-SUMO, pET28a-csgA-SUMO-SmtA, pET28a-csgA-SmtA, pET28a-csgA-SUMO-shMT, pET28a-csgA-shMT. (FG, QG)

2022/9/9-16
Biofilm growth within MBBR carrier K1. (AH)

2022/9/14
Culturing E. coli shMt, SUMO-shMt, SUMO-SmtA and SmtA with heavy metal ions (Cu(II), Cd(II)). (FG, QG)

2022/9/17
Same as 2022/8/22. (AH, WC)

2022/9/21
Conducting microwave digestion on samples obtained at 2022/9/17 to. (AH, WC)

2022/9/22
Conducting ICP-MS on samples obtained at 2022/9/14 to test silver absorption by bacteria. (AH, WC)

2022/9/23
Conducting ICP-MS to test the adsorption of copper and cadmium by the bacteria (2022/9/22 digested samples). (AH, FG, QG, WC)

2022/9/28-10/3
Biofilm growth and measurement. (AH)

Part 0 overview

2022/3-2022/10
1.Weekly Meeting regarding comprehensive progression with members 2022/3-2022/10
2.Regularly Meeting with PI and domain experts

3.Meeting and collaboration with other teams regarding Model session (DKU_China,NJU,JPU,CPU,NPU,etc)

Part 1 Molecular Model (JL)

2022/3-2022/5
1.Brainstroming, Training on system biology,data science,ML/DL,Matlab/Python/R/shell,etc.

2022/6
2.Computational framework for linker design, Alphafold2 structure prediction

2022/7
3.Formulate the idea of Computational approach Molecular dynamics to assist Design- Build-Test-Learn of our Bioremedation system

2022/8
4.Run Gromacs Molecular dynamic simulation on XJTLU bioinformatics lab Linux server and relevant results visualization; Run Autodock vina on loacal windows virtual machine(View detailed documentation)

2022/9
5.Results discussion and wiki writing

Part 2 biological pathway (WL)

2022/3-2022/5
1.Research on the mechanism of the absorption of silver ions and the detection mechanism of the CusS-CusR(Two-Component System, delete at last).
2.read articles about the values of some key parameters and consider how other team or people create a model

2022/6
1.Build the model of Two-Component system including the transport of the metal ions and made assumptions(deleted at last)
2.Build the model of the absorption of AgNP by MBP
3.Talk with experimental group about the design of experiment so that key parameters can be obtained

2022/7
1,redesigned the model of the absorption of AgNP
2,tried to research more deeply the growth of CsgA and tried to use the concentration of amino acid to represent the maximal concentration of the CsgA(inefficient and impractical)

2022/8
1,Learnt about the mechanism of the absorption by MT and built the model about it
2, Theeffect of sumo
3, Fit the experimental data

2022/9
1, Built wiki

Part 3 Survival Analysis (JLU)

2022/3-2022/5
1.Brainstorming, preparing for data processing, ML/DL, modeling method, synthetic biology, Python/R,etc.

2022/6
Develop a quantified three-state transition growth model. Evaluate the biological processes of population survival parametrically.

2022/7
Perform dependence analysis of the parameters in model and study how these parameters change with increasing metal ion concentrations. Also, evaluate the effect of our genetic modification on the bacterial tolerance to metal ions.

2022/8
Establish network of relationships between heavy metal ion concentrations and the corresponding survival parameters. Predict the survival of E. coli in real wastewater environments and provide guidance for practical operations.

2022/9
Logic sorting, results disscussion and wiki writing.

Part 4 Fluid mechanics (JL, JLU, WL)

2022/7
1,Considered the diffusion in the device, especially the effect of MBBR
2,Learn about the details about diffusion equation, Narrer-stoke equation and so on.

2022/8
1,Tried to solve the diffusion equation with matlab
2,Considered how to express the output concentration of metal ions in a meaningful way

2022/9
1, Built wiki
2, Have a meeting with Nanjing Foreign Language School

2022/3 (BC, QZ, WC)
Follow news and current affairs to find local problems.

2022/4/30 (All students)
Due to the pandemic, we were in home quarantine until June. During this period, we discovered the problem of water pollution and consulted relevant literature on sewage treatment, and gained an intuitive understanding of the problem.

2022/5/12 (QZ, BC)
We went to Green Jiangnan Public Welfare Organization for interview.

2022/5/30 (QZ, BC)
We sorted out the literature we read and formulated the project plan and feasibility report based on the experimental design.

2022/6/12 (All students)
We were allowed to return to school for offline research and social practice.

2022/6/17 (All students)
We went to Shanghai Cheng Feng Technology Corporation and discussed with the director.

2022/6/29 (All students)
We went to sewage plant in the new district of Suzhou to have a further understanding of the water treatment process.

2022/7/1 (All students)
We handed out the questionnaires. Meetup with DKU.

2022/7/5 (QZ, BC)
Meetup with CAU.

2022/7/4 (QZ, BC, WC)
Meetup with SHTU.

2022/7/10 (QZ, BC)
We analyzed the data from questionnaires.

2022/7/13 (QZ, BC, WC)
Meetup with NJU.

2022/7/14 (QZ, WC)
we planned a face-to-face meeting with experts in biofilm field to understand biofilm in practice.

2022/7/16 (QZ, BC, WC)
Meetup with multiple teams.

2022/7/18 (QZ, BC, WC)
Meetup with GXU-China.

2022/7/20 (QZ, BC, WC)
We started to design the prototype of the hardware. Meetup with JLU.

2022/8 (QZ, BC)
We consulted with experts in environmental science and biological science for suggestions on the project.

2022/9 (All students)
We reflected on some problems about surveillance and biosaty issues.