Team:ZJU-China/Notebook
Notebook
Brain storm
In March and April, we held a meeting every third days to discuss what question were to be addressed in this research. Each member of our team was required to go through 20+ literature and/or wiki pages of iGEM teams before 2021. We added our ideas to candidate project title list and then delete ideas that violates basic rules of a good research (i.e. creativity, academic importance, and feasibility).
March 6
The kick-off meeting of ZJU-China 2021
Time: 19:00—20:00, March 6
Location: Room 234, College of Life Science, Zijin'gang Campus, Zhejiang University, China
Recorder: Wu Hao-Ran
Highlights:
- Self-introduction
- About iGEM competition: Track list, the iGEM cycle (i.e. timeline), deliverables and due date in 2020, judging handbook, medals & final lists & special prizes
- Team culture
March 10
Time: 21:10—23:30, March 10
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Mao Zhuo
Highlights:
Brief instructions by Liang Qi-Yu, the leader of team ZJU-China 2020
An overview of human practice, collaboration, measurement, and improvement
Ideas of project design in Week 1:
- Huang Guan-Rui: Sigma Pathway – Cell-based signal pathway modules
- Huang Guan-Rui: Programming bacteriophage using yeast
- Lei Jiang-Chu: Reconstruct dental calculus based on gram-negative bacteria
- Lei Jiang-Chu: Hemocyte counting using bone marrow aspiration
- Yang Lin-He: Constructing lactic acid bacteria for manufacturing pickle
- Yang Lin-He: Targeted therapy of cancer based on engineered bacteria
- Yang Lin-He: Liver cancer therapy using engineered virus
- Wu Hao-Ran: Remote sensing in engineered bacteria based on optogenetics
- Cheng Ji-Ou: Sequestration of heavy metals in soil using microbial community
- Wang Yong-Yin: Prevention of postharvest disease in bayberry using biofilm
- Wang Yong-Yin: Construction of protein transfer bacteria based on sulfate
- Hu Long-Shuang: Construction of iVAX vaccine
- Hu Long-Shuang: Delimitation of fishy odor using engineered bacteria
March 13
Time: 21.10—23:00, March 13
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Cheng Ji-Ou
Highlights:
Lab safety training by Yang Fan (Secondary PI, team ZJU-China 2021)
Project timeline in brain storm stage
Discuss the existing ideas in Week 1: We assessed the academic importance, social values, and feasibility of each project design. After filtering, only three ideas remained on the list
- Huang Guan-Rui: Sigma Pathway – Cell-based signal pathway modules #1
- Lei Jiang-Chu: Reconstruct dental calculus based on gram-negative bacteria #2
- Yang Lin-He: Liver cancer therapy using engineered virus #3
Task in the next week: Experimental design of each idea
- #1: Huang Guan-Rui, Mao Zhuo, and Yang Lin-He
- #2: Lei Jiang-Chu, Hu Long-Shuang, Zhang Bo-Yan, and Wang Yong-Yin
- #3: Lei Jiang-Chu and Yang Lin-He
- New ideas: Cheng Ji-Ou and Wu Hao-Ran
Lei Jiang-Chu: Design of human practice work
March 17
Time: 19:00—21:00, March 17
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Hu Long-Shuang
Highlights:
Assess candidate ideas:
- #1: Sigma Pathway – Cell-based signal pathway modules. Separating pathways in various chambers add complexity of cell communication. Further improvement includes design of modular pathways similar to protein-level regulation. Logic gate at cell-level is considered in project design. Optogenetics can be integrated into the construction of cell pathways.
- #3: Liver cancer therapy using engineered virus. Virus engineering can be broken into four parts: targeted infection, reproduction, elimination of cancer cells, and avoidance of immune clearance.
New ideas: protein evolution, optogenetics oscillator, myxomycete, etc.
March 20
Time: 21:10—23:00, March 20
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Huang Guan-Rui
Highlights:
Discuss the feasibility of existing project designs. Only #3 remains. We decided to use adenovirus as our chassis organism. Yang Lin-He provided possible solutions relevant to potential problems in the project design (e.g., improve specificity, safety, etc.).
New ideas: killing mosquitoes, hair dye cosmetics, microbe illustrations, elimination of disease in grape caused by continuous cropping
Task in the next week: Experimental design of each idea
- #3: Liver cancer therapy using engineered virus – Lei Jiang-Chu, Yang Lin-He, and Huang Guan-Rui
- #4: Microbe illustrations (NEW) – Huang Guan-Rui, Cheng Ji-Ou, and Che Zi-Yuan
- #5: Disease in grape (NEW) – Mao Zhuo, Hu Long-Shuang, Wang Yong-Ying, and Zhang Bo-Yan
Experimental design of Measurement
March 24
Time: 19:00—21:00, March 24
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Lei Jiang-Chu
Highlights:
Partnership: NJU-China 2021
Improvements on each project design (i.e. #3, #4, and #5)
March 27
Time: 21:10—23:00, March 27
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Wang Yong-Ying
Highlights:
Measurement: Some candidate parts are BBa_I746365 and BBa_I765001. We also decided to investigate the efficiency of expressing GFP induced by IPTG and various types of metal ions.
Further discussion about existing project designs
Safety consideration
March 31
Time: 19.00—21:00, March 31
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijingang Campus, Zhejiang University, China
Recorder: Wu Hao-Ran
Highlights:
Measurement: Part BBa_K540001 was chosen for improvement.
Further discussion about existing project designs
New ideas: Elimination of fire ant, game of cell battle.
Task in the next week: Experimental design of each idea
- Diseases in grape & earthworm: Wang Yong-Ying, Mao Zhuo, Zhang Bo-Yan, and Cheng Ji-Ou
- VirusGuard (#3): Lei Jiang-Chu, Yang Lin-He, Huang Guan-Rui, Hu Long-Shuang
- Fire ant: Zhang Bo-Yan, Wu Hao-Ran
- Game of cell battle: Huang Guan-Rui
April 5
Time: 21:10—23:00, April 5
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Huang Guan-Rui
Highlights:
- Measurement: Co2+ was chosen for part characterization
- Further discussion about existing project designs: Biological characteristics of fire ant and earthworm.
April 7
Time: 19:00—21:00, April 7
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Yang Lin-He
Highlights:
- Measurement: design of improved parts using T7 promotor
- Further discussion about existing project designs: Due to unaddressed safety and technical problems in some project design (e.g. diseases in grape, degradation of plastics using earthworm, only VirusGuard and Fire Ant project were kept.
April 10
Time: 21:10—23:00, April 10
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Zhang Bo-Yan
Highlights: Further discussion about existing project designs – VirusGuard, Diseases in grape, and Fire Ant
April 14
Time: 19:00—21:00, April 14
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Mao Zhuo
Highlights:
- We focused on the last two candidate project design (i.e. VirusGuard and Fire Ant)
- VirusGuard: Get access to P2 laboratory, lab safety
May 5
The close-out meeting of brain storm stage
Time: 19:00—21:00, May 5
Location: Room 413, National Demonstration Center for Experiment Biology Education (ZJU), Zijin'gang Campus, Zhejiang University, China
Recorder: Wu Hao-ran
Highlights:
After an exhausting work of literature search and exam block in our university, we ended brain storm stage by finalizing our project title
Project VirusGuard won the vote against project Fire Ant at 8:5
Group members are divided into four parts based on project design:
- Targeted infection: Yang Lin-He*, Wu Hao-Ran
- Targeted elimination of liver cancer cells: Huang Guan-Rui*, Zhang Bo-Yan
- Virus reproduction with specificity: Lei Jiang-Chu*, Mao Zhuo, and Wang Yong-Ying
- Avoidance of immune clearance: Hu Long-Shuang*, Cheng Ji-Ou
Where ‘*’ donates to leader of each subgroup.
Experiments
The second stage of our research started in May 5, the day we finalized our brainstorm ideas – VirusGuard. We divided members in wet lab into four subgroups, which is consist with the project design.
Targeted Infection
5.19
– Pre-experiment Design: Express scfv using Shuffle T7-K12 competent cell
6.9 – 6.14
– Wu Hao-Ran: Transformation of scfv-pET28 plasmid into shuffle T7 competent cell. IPTG induction for 16h.
– Yang Lin-He: Protein expression of Rosetta strain
6.15 – 6.20
– Wu Hao-Ran: Protein expression of scfv-pET28-shuffle T7 strain (SDS-PAGE). No band was detected. We postulated that high temperature might reduce the efficiency of protein expression
– Wu Hao-Ran: Plasmid extraction of scfv-pET28-shuffle; Electrophoresis
6.21 – 7.2
– Yang Lin-He: Protein purification of Rosetta-scfv strain
– Wu Hao-Ran: IPTG induction experiment of scfv-pET28-shuffle T7 strain
7.2 – 7.8
– Yang Lin-He & Wu Hao-Ran: Coomassie brilliant blue staining & Western Blot
– Yang Lin-He & Wu Hao-Ran: IPTG induction experiment of scfv-pET28-shuffle T7 strain. Gradient: IPTG – 0, 0.05, 0.1M; Temperature: 10℃, 12.5℃, 15℃, and 17.5℃. Cell disruption was followed.
7.9 – 7.12
– Yang Lin-He & Wu Hao-Ran: Coomassie brilliant blue staining & Western Blot. We found that IPTG induction at low temperature was of high efficiency.
– Yang Lin-He & Wu Hao-Ran: Western blot of IPTG-induced scfv expression at low temperature; Cell disruption of various types of samples used as antibody.
7.13 – 7.14
– Yang Lin-He & Wu Hao-Ran: Mass induction (200ml) at 10℃
7.15 – 7.17
– Yang Lin-He: Cell disruption, protein purification, coomassie brilliant blue staining, and western blot
7.16 – 7.19
– Yang Lin-He: Mass induction (800ml). In 7.19, the results showed that the intensity of protein in supernatant was higher than that in the sediment
7.20 – 7.23
– Wu Hao-Ran: Culturing shuffle T7 strain. Detection of protein expression in various types of cell samples
7.23 – 25
– Yang Lin-He: Western-blot, coomassie brilliant blue staining, and purification
– Yang Lin-He & Wu Hao-Ran: Protein purification & Dialysis
7.28
– Yang Lin-He: Mass induction (200ml ×4) of Rosetta-RS strain at 10℃
7.29 – 30
– Yang Lin-He: Western blot of protein sample after dialysis & supernatant in temperature-gradient experiments
8.1 – 8.3
– Zhang Bo-Yan & Wu Hao-Ran: Cell disruption, coomassie brilliant blue staining, and western blot in mass induction experiments in 7.28
– Wu Hao-Ran: Mass IPTG induction of scfv-RS strain (100ml ×4) with IPTG gradient (1:50, 1:100, 1:200, and 1:1000). No significant difference of the intensity of protein expression was detected
8.3 – 8.5
– Wu Hao-Ran: Mass IPTG induction of scfv-RS strain (100ml ×4) with temperature gradient (10℃, 20℃, 30℃, and 37℃)
Targeted elimination
5.9
– Experimental Design: Contact companies to synthesis siRNA and then conduct transfection
– Pre-experiment Design: Expressing siRNA in bacteria. Validation techniques include electrophoresis and/or rt-PCR
– Experimental Design: Use GFP to characterize efficiency of expressing siRNA
5.19
– Reagent Preparation/buying: Design of siRNA primer, pCMV (GV514), pU6 (GV112) plasmid
6.9 – 6.14
– Zhang Bo-Yan: Construction of shRNA vector (failed)
6.15 – 6.20
– Zhang Bo-Yan: ligation and transformation of plasmid
– Huang Guan-Rui: Construction of shRNA vector (failed)
6.21 – 7.6
– Exhausted work – Construction of shRNA (both by Huang Guan-Rui and company)
7.6 – 7.8
– Huang Guan-Rui: Successful construction of shRNA vector; sequencing
7.9 – 7.12
– Validation of shRNA vector using sequencing (by company)
7.12 – 7.15
– Huang Guan-Rui: Construction of remaining shRNA vectors
7.15 – 7.18
– Huang Guan-Rui: Culture Hek293t; transfection of shRNA vector
7.18 – 7.21
– Huang Guan-Rui: Western blot. The results showed that shRNA vector constructed by company was effective
7.22 – 7.25
– Huang Guan-Rui & Zhang Bo-Yan: Culture Hek293t; Transfection
7.26 – 7.29
- Huang Guan-Rui: Western blot. The result is not perfect which may due to over-transfaction
7.30 – 8.2
- Huang Guan-Rui & Zhang Bo-Yan: Measuring the expression efficiency of promotors
8.3 – 8.5
- Zhang Bo-Yan: Transfaction
8.6 – 8.8
- Zhang Bo-Yan: Western blot. The result is not good. Some problems may occur and need further analysis
8.9 – 8.15
- Huang Guan-Rui & Zhang Bo-Yan: Western blot, transfaction, and qRT-PCR
9.6 – 9.9
- Zhang Bo-Yan: qRT-PCR
Virus reproduction
5.9
– Experimental Design: Insert miRNA to 3'UTR sequence using homologous recombination method
– Experimental Design: Insert sponge sequence after cancer-specific promotor
5.19
– Synthesis of sponge sequence
6.9 – 6.14
– Lei Jiang-Chu & Wang Yong-Yin: Construction of zeroload plasmid, transfection, and sequencing
– Lei Jiang-Chu: bacteria liquid PCR of complement plasmid
– Lei Jiang-Chu: Passage of HepG2
– Wang Yong-Yin: PCR and electrophoresis of zeroload plasmid
– Mao Zhuo: PNK and ligation of complement plasmid
6.17 – 6.20
– Wang Yong-Yin & Lei Jiang Chu: Plasmid linearization and purification
– Mao Zhuo: Sequencing results of complement plasmid showed that construction failed. Electrophoresis of zeroload plasmid succeeded but could not be sequenced due to low concentration
7.7 – 7.12
– Wang Yong-Yin: Successful construction of 3' UTR and Sponge vector
– Lei Jiang-Chu: E. coli that contained complement plasmid formed colony
– Bought cell culture plate
7.12 – 7.18
– Wang Yong-Yin: Sequencing of complement plasmid
7.19 – 7.31
– Mao Zhuo: Pre-experiment of PBMC, 293, HepG2 – qRT-PCR. Explore optimal concentration of template in reverse transcription
– Mao Zhuo: Successful transfection of 3U, sponge plasmid
– Mao Zhuo: Transfection of 3U, 3U zeroload plasmid, 3U+sh199, and 3U zeroload+sh199 plasmid
– Mao Zhuo: synthesis of shRNA complement plasmid (by company)
8.1 – 8.5
– Wang Yong-Yin: Sequencing empty sponge; qRT-PCR
– Wang Yong-Yin: Reconstruction of 3U empty plasmid
8.6 – 8.8
– Wang Yong-Yin: qRT-PCR
– Wang Yong-Yin: transduction of 3U+sh199
8.9 – 8.15
– Wang Yong-Yin: Design and validation of new primer in qRT-PCR expeirment
8.19
– Mao Zhuo: Transfaction of hepG2 using 3U empty plasmid
– Mao Zhuo: Transduction of miRNA 195 and 22.
8.22
– Mao Zhuo: Functional analysis of miRNA 199
– Mao Zhuo: Functional analysis of 3U plasmid
– Wang Yong-Yin: qRT-PCR
8.23 – 9.4
– Wang Yong-Yin: qRT-PCR
9.6
– Wang Yong-Yin: Complete all experiments
Immune escape
5.9
– Experimental Design: Insert TLR9i to 3'UTR sequence using homologous recombination method (or genome-wide PCR)
– Experimental Design: Transfection into cells and then detect IL-12 using rt-PCR
5.19
– Reagent Preparation/buying: ODN 2006 (ODN 7909), agonist, antagonist, HepG2 cell
6.9 – 6.16
– Cheng Ji-Ou: Bacteria liquid PCR
– Hu Long-Shuang: Electrophoresis
6.17
– Cheng Ji-Ou: Electrophoresis
– Yang Lin-He: Cell preservation
6.18
– Sequencing (by company)
6.19
– Hu Long-Shuang & Cheng Ji-Ou: Learnt transfection
6.20
– Hu Long-Shuang & Cheng Ji-Ou: Ligation
– Hu Long-Shuang & Cheng Ji-Ou: Learnt PMBC cell culture
– Yang Lin-He, Hu Long Shuang & Cheng Ji-Ou: Passage of PBMC
6.22
– Cheng Ji-Ou: Ligation and transformation
6.23
– Bacteria liquid PCR
– Sequencing (by company)
7.14
– Cheng Ji-Ou: Purification, ligation, and transformation
7.17 – 7.18
– Bacteria liquid PCR and sequencing
7.23
– 2216-TLR9i plasmid construction completed by company
7.24
– Hu Long-Shuang: Transformation
– Cheng Ji-Ou: PBMC cell thawing
7.26 – 7.30
– Cheng Ji-Ou: cell inheritance
– Cheng Ji-Ou: Transfaction
7.31 – 8.1
– Hu Long-Shuang: Collect the supernatant
8.3 – 8.12
– Hu Long-Shuang & Cheng Ji-Ou: ELISA
8.15 – 8.16
– Cheng Ji-Ou: Culturing PBMC cells
8.18
– Cheng Ji-Ou: Transfaction of PBMC
9.17 – 9.20
– Hu Long-Shuang & Cheng Ji-Ou: ODN stimulation, ELISA, and transfaction
9.21 – 9.26
– Hu Long-Shuang & Cheng Ji-Ou: Repeat transfaction experiment
Measurement
5.27
– Construction and validation of sPrcn Promotor (sequencing by company)
6.9 – 6.14
– Yang Lin-He & Wu Hao-Ran: IPTG induction in Prcn and sPrcn
– Yang Lin-He & Wu Hao-Ran: Quantitative detection of GFP expression in Prcn and sPrcn with various gradient (i.e. induction time, concentration of IPTG, and concentration of heavy ions)
– Wu Hao-Ran: Statistical analysis
6.15 – 6.20
– Wu Hao-Ran: 2nd induction experiment of Prcn and sPrcn
– Wu Hao-Ran: Statistical analysis. We found that high concentration of Co2+ might kill engineered cells.
6.21 – 7.8
– Wu Hao-Ran: 3rd induction experiment of Prcn with lower concentration of Co2+
– Yang Lin-He: Induction of sPrcn with Co2+ or Ni2+
7.14 – 7.18
– Wu Hao-Ran: 4st induction experiment of Prcn with various types of metal ions and time gradient.
– Wu Hao-Ran: Induction experiment of sPrcn
– Wu Hao-Ran: Data analysis
Wiki
The third stage of our project lies in the September and October. Our wet lab had gotten most of the results and our dry lab has completed most of the work. In late August, we started to write our wiki although we did a lot of revision work in September and October.
We gained the rest of our experimental result in Septemper and early October. So a little bit revision on our Experiment and Result page is conducted.
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