iGEM 2021 — Team ZJU-China

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# Team:ZJU-China/Notebook

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* Team

  [Team](https://2021.igem.org/Team%3AZJU-China/Team)
  [Attributions](https://2021.igem.org/Team%3AZJU-China/Attributions)
* Project

  [Description](https://2021.igem.org/Team%3AZJU-China/Description)
  [Design](https://2021.igem.org/Team%3AZJU-China/Oursolution)
  [Engineering](https://2021.igem.org/Team%3AZJU-China/Engineering)
  [Experiments](https://2021.igem.org/Team%3AZJU-China/Experiments)[Contribution](https://2021.igem.org/Team%3AZJU-China/Contributions)[Notebook](https://2021.igem.org/Team%3AZJU-China/Notebook)
  [Proof of Concept](https://2021.igem.org/Team%3AZJU-China/Proof_Of_Concept)
  [Results](https://2021.igem.org/Team%3AZJU-China/Results)
* Parts

  [Part Collection](https://2021.igem.org/Team%3AZJU-China/Part_Collection)
  [New Parts](https://2021.igem.org/Team%3AZJU-China/Parts)
  [Improve](https://2021.igem.org/Team%3AZJU-China/Improve)
* [Safety](https://2021.igem.org/Team%3AZJU-China/Safety)
* Human Practices

  [Integrated HP](https://2021.igem.org/Team%3AZJU-China/Integrated_Human_Practice)
  [Human Practice](https://2021.igem.org/Team%3AZJU-China/Human_Practices)
  [Collaboration](https://2021.igem.org/Team%3AZJU-China/Collaborations)
  [Partnership](https://2021.igem.org/Team%3AZJU-China/Partnership)
  [Public engagement](https://2021.igem.org/Team%3AZJU-China/Education)
  [Inclusivity](https://2021.igem.org/Team%3AZJU-China/Inclusivity)
  [Implementation](https://2021.igem.org/Team%3AZJU-China/Implementation)
  [Communication](https://2021.igem.org/Team%3AZJU-China/Communication)
* DRY LAB

  [Hardware](https://2021.igem.org/Team%3AZJU-China/Hardware)
  [Measurement](https://2021.igem.org/Team%3AZJU-China/Measurement)
  [Model](https://2021.igem.org/Team%3AZJU-China/Model)
  [Software](https://2021.igem.org/Team%3AZJU-China/Software)
* [Medals](https://2021.igem.org/Team%3AZJU-China/Medals)

Notebook

* [Brain storm](#BrainStorm)
* [Experiments](#Experiments)
* [Wiki](#Wiki)

# 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.

![](https://static.igem.org/mediawiki/2021/7/7e/T--ZJU-China--myfooter.png)

# FOLLOW US

[![](https://static.igem.org/mediawiki/2021/7/70/T--ZJU-China--Bilibili.png)](https://space.bilibili.com/62518664)
[![](https://static.igem.org/mediawiki/2021/1/1e/T--ZJU-China--Twitter.png)](https://twitter.com/ZjuChina)
[![](https://static.igem.org/mediawiki/2021/b/b7/T--ZJU-China--Wechat.png)](https://mp.weixin.qq.com/s/0PDkLfb3hTimV7LSZ9Ayew)

# CONTACT US

- ZJU\_China@outlook.com
Hangzhou 310058
866 Yuhangtang Rd
P.R. China

ZJU-China

ZJU-China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:ZJU-China/Engineering

[![](https://static.igem.org/mediawiki/2021/d/d0/T--ZJU-China--newlogowhite.png)](https://2021.igem.org/Team%3AZJU-China)

Menu

* Team

  [Team](https://2021.igem.org/Team%3AZJU-China/Team)
  [Attributions](https://2021.igem.org/Team%3AZJU-China/Attributions)
* Project

  [Description](https://2021.igem.org/Team%3AZJU-China/Description)
  [Design](https://2021.igem.org/Team%3AZJU-China/Oursolution)
  [Engineering](https://2021.igem.org/Team%3AZJU-China/Engineering)
  [Experiments](https://2021.igem.org/Team%3AZJU-China/Experiments)[Contribution](https://2021.igem.org/Team%3AZJU-China/Contributions)[Notebook](https://2021.igem.org/Team%3AZJU-China/Notebook)
  [Proof of Concept](https://2021.igem.org/Team%3AZJU-China/Proof_Of_Concept)
  [Results](https://2021.igem.org/Team%3AZJU-China/Results)
* Parts

  [Part Collection](https://2021.igem.org/Team%3AZJU-China/Part_Collection)
  [New Parts](https://2021.igem.org/Team%3AZJU-China/Parts)
  [Improve](https://2021.igem.org/Team%3AZJU-China/Improve)
* [Safety](https://2021.igem.org/Team%3AZJU-China/Safety)
* Human Practices

  [Integrated HP](https://2021.igem.org/Team%3AZJU-China/Integrated_Human_Practice)
  [Human Practice](https://2021.igem.org/Team%3AZJU-China/Human_Practices)
  [Collaboration](https://2021.igem.org/Team%3AZJU-China/Collaborations)
  [Partnership](https://2021.igem.org/Team%3AZJU-China/Partnership)
  [Public engagement](https://2021.igem.org/Team%3AZJU-China/Education)
  [Inclusivity](https://2021.igem.org/Team%3AZJU-China/Inclusivity)
  [Implementation](https://2021.igem.org/Team%3AZJU-China/Implementation)
  [Communication](https://2021.igem.org/Team%3AZJU-China/Communication)
* DRY LAB

  [Hardware](https://2021.igem.org/Team%3AZJU-China/Hardware)
  [Measurement](https://2021.igem.org/Team%3AZJU-China/Measurement)
  [Model](https://2021.igem.org/Team%3AZJU-China/Model)
  [Software](https://2021.igem.org/Team%3AZJU-China/Software)
* [Medals](https://2021.igem.org/Team%3AZJU-China/Medals)

On this page, we introduce the engineering design cycle of how we developed Viruguard in chronological order. We made four rounds of improvement to Viruguard before we ultimately got a mature Viruguard product. We firstly constructed its virulence to cancer cells and then tried to shield normal cells from its virulence. Finally, an oncolytic virus, Viruguard, was produced, which had a high level of security and virulence respectively to normal cells and cancer cells.

Engineering success

* [The enginnering cycle](#cycle)* [ViruGuard Development](#Improvement)
  * [References](#ref)

# The enginnering cycle

![](https://static.igem.org/mediawiki/2021/f/f9/T--ZJU-China--Engneering-Cycle_final.png)

**Synthetic biology** is a subject that differs a lot from the conventional ones. In traditional life science subjects, researchers are supposed to use the scientific method to obtain accurate results, whose work mainly focused on making observations and performing experiments. However, synthetic biology goes
beyond that, it is all about constructing and innovating a composite system from different parts and biobricks. Due to the distinctiveness of Synbio, an engineering cycle should be established from the beginning and undergoes continuous improvement with the results of experiments discoverd, which would finally give birth to a successful engineered product, namely a genetically engineered machine. And that was why we documented our engineering below which had helped us perfect Viruguard throughout the whole iGEM competition this year.

# ViruGuard Development

![](https://static.igem.org/mediawiki/2021/0/07/T--ZJU-China--Engneering-Circle1.png)

## **Research 1:** *How can we use organism to kill the tumor?*

In current hepatocellular carcinoma guidelines, traditional treatment including **surgery, chemotherapy, and radiotherapy** are still recommended more often than novel treatment. These methods directly eliminate or shrink the tumor in situ, and even with the development of medicine, limited changes could be made to these conventional therapies in a short term. In synthetic biology,
however, we can engineer organisms according to our exact needs, which in this case, means HCC treatment.
Thus, we set out to explore the appropriate **viruses** that possess the ability to infect cells and **prokaryotic
bacteria**, which are widely used in synthetic biology, and could make it easier to conduct following experiments.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/5/59/T--ZJU-China--Engneering-Circle2.png)

## **Imagine 1:** *Which type of organism is better for the project?*

At the very first, we couldn't make our mind on what kind of virus to choose as our chassis organism.

Generally, we focused on the most normally used organism, *Escherichia Coli*, to be the possible chassis organism. As it has already been deeply and widely studied before, it might be easy for us to conduct transformation on the bacteria, and shape it according to our expectations. Besides, as *E.coli* couldn't infect human cells directly,
the risk of safety incidence is relatively low. However, several problems had made us give up the idea. The difficulty of turning it into a eukaryon-infectable type of bacteria and the uncertainty of whether the procaryotic gene can function in the eukaryotes were the most challenging problems that we were incapable to solve.

Therefore, we broadened our vision and put the attention to the virus. Because of its natural characteristics, the viral gene could express in human cells, and there have already been some literature supporting the feasibility of virus treatment. Nevertheless, in previous studies, although researchers have extracted virus strains with relatively weak toxicity, current virus treatment still could pose **grievous threats** to animal models in experiments or patients in the clinical trials. So this is the first problem that we need to work out.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/ee/T--ZJU-China--Engneering-Pic3.png)

## **Design 1:** *How can we ensure Viruguard to kill cancer cells in a safe way?*

**The main focus of our design is on keeping the virulence of virus to kill malignant cells in a safe way.**
We had thought of making our mechanism in a trascriptional, translational or epigenetical level and finally
we determined that the best solution of problem is using a peculiar promoter to regulate the expression of
**gene-targeting siRNA**. After we had a deeper understanding to
the RNAi mechanism, we noticed that it would not be functional if we merely tried to express the
siRNA sequence. Thus, we expressed the shRNAs in the cells which would further be processed into the
mature siRNAs.
Here are some results of function test of our designed shRNAs. From BBa\_K3730023 to BBa\_K3730030, they are all our shRNA primers used for RNAi killing mechanism.

#### [**BBa\_K3730023**](http://parts.igem.org/Part%3ABBa_K3730023) [**BBa\_K3730024**](http://parts.igem.org/Part%3ABBa_K3730024)

#### [**BBa\_K3730025**](http://parts.igem.org/Part%3ABBa_K3730025) [**BBa\_K3730026**](http://parts.igem.org/Part%3ABBa_K3730026)

![](https://static.igem.org/mediawiki/2021/1/12/T--ZJU-China--Engneering-pic1.png)

**Fig.1** Two targeting gene for tumor survival suppressing.Left: hTERT, human transcriptase gene; Right: VEGFA, vascular endothelial growth factor A[1].

To guarantee the security of our design, we decided to use **tumor specific promoter** to regulate the transcription of our designed shRNA sequence. We chose ***phTERT*** and ***psurvivin*** as the promoter to shoulder this responsibility because they have significant activity difference in the normal and cancer cells.

#### hTERT promoter Sequence 263bp

CAGCTGCGCTGTCGGGGCCAGGCCGGGCTCCCAGTGGATTCGCGGGCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTTTCAGGCAGC

#### [**BBa\_K3730017**](http://parts.igem.org/Part%3ABBa_K3730017)

#### survivin promoter Sequence 397bp

ATTACAGGCATGCACCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAAGGTTTCACCGTGATGGCCAGGCTGGTCTTGAACTCCAGGACTCAAGTGATGCTCCTGCCTAGGCCTCTCAAAGTGTTGGGATTACAGGCGTGAGCCACTGCACCCGGCCTGCACGCGTTCTTTGAAAGCAGTCGAGGGGGCGCTAGGTGTGGGCAGGGACGAGCTGGCGCGGCGTCGCTGGGTGCACCGCGACCACGGGCAGAGCCACGCGGCGGGAGGACTACAACTCCCGGCACACCCCGCGCCGCCCCGCCTCTACTCCCAGAAGGCCGCGGGGGGTGGACCGCCTAAGAGGGCGTGCGCTCCCGACATGCCCCGCGGCGCGCCATTAACCGCCAGATT

#### [**BBa\_K3730018**](http://parts.igem.org/Part%3ABBa_K3730018)

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/e7/T--ZJU-China--Engneering-Pic4.png)

## **Build 1:** *How can we build it?*

After we assessed the difficulty of directly constructing a plasmid containing the tumor specific promoters and shRNA simultaneously, we decided to seperate them into two vectors to obtain a higher operability in our experiments, but still in the final virus package, shRNA would be put under the control of tumor specific promoter. We are going to build two vectors: the tumor specific promoter vector and the shRNA expression vector. We got the full sequence of *htert* and *survivin* promoter from
human genome by PCR and replaced the original promoter sequence in pEGFP-C1 with the sequence that we got. As for the shRNA expression vector, we found it was hard to construct because of the difficulty of integrating the short sequence into the vector. Therefore, we made the decision to sythesize it directly as clonal plasmids.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/7/7d/T--ZJU-China--Engneering-Cycle5.png)

## **Test 1:** *Do we get what we want?*

The result satisfied our expectations generally. We transfected shRNA plasmids into HepG2 cells and **analyzed the apoptosis rate** to check the efficiency of the killing mechanism. The mRNA and protein expression level of *vegfa* and *htert* showed a decline in the context of shRNA that we designed. We also witnessed **a dominant increase of induced apoptosis** in shRNA groups by using the Annexin kit. These facts all showed that the shRNA played a role in inducing the apoptosis of HepG2. We transfected vectors containing two types of promoters into HepG2 and HEK293T cells. We observed that the fluorescence intensity is much higher in HepG2, **indicating that these two promoters had an activity difference in normal and tumor cells**.

![](https://static.igem.org/mediawiki/2021/1/17/T--ZJU-China--Engneering_pic3.png)

**Fig.2** Expression Level of promoter in two different types of cells.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/5/56/T--ZJU-China--Engneering-Cycle6.png)

## **Learn 1:** *What we did not know about our design?*

**As the mechanism we focused on was related to the HepG2 cell line and its specific growth characteristic, *in vitro* experiments was not enough to illustrate its killing efficacy to tumor *in vivo*.** VEGFA is responsible for angiogenesis, which plays important role in the nutrition supply to tumor in situ, therefore the change led by anti-VEGFA shRNA couldn't be witnessed in culture dish. hTERT is responsible for the infinite proliferation ability of cancer cells. Though we had seen the increasing apoptosis rate in cell culture plate environment, the result was not convincing enough to demonstrate its function in human body. **We will still need further *in vivo* experiments to confirm shRNA clinical function which may cause severe biosafety accident on current stage.**

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/8/8c/T--ZJU-China--Engneering-Cycle7.png)

## **Improve 1:** *Is it enough for our design?*

The first version of ViruGuard had just realized the expectation to be able to kill the tumor cell and it's safe to some extent. However, the result had shown that in normal cell situations, the promoter is still expressed at a relatively low level. There was still the possibility of potent risk to normal cells. Therefore, we had to continue to put restrictions on the killing mechanism activation. Therefore, we had to think about other ways to **build a specific expression mechanism to reduce the risk.**

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/0/07/T--ZJU-China--Engneering-Circle1.png)

## **Research 2:** *Another possible mechanism for improving specificity.*

The way that we designed in the killing mechanism to limit the virulence was not enough for a
treatment that would be possibly used for real patients. We must find other ways to make the
virus distinguish the normal from the tumor cells. Therefore, we tried to find some other differences between
normal and HCC cells. We mainly focused on the different expression level of proteins and cytokines in these two
cell types to see whether their expression differences can be used to set up a second fence to protect normal cells
from the virulence of Viruguard.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/5/59/T--ZJU-China--Engneering-Circle2.png)

## **Imagine 2:** *Which type of "difference" can be used?*

During the process that we tried to program the Viruguard to make it distinguish the normal cells and malignant cells we mainly focused the content in the cytoplasm including protein, cytokine
and miRNA. We first tried to use protein and cytokine to help because of their essential role in
regulating cell function. However, we realized that this could hardly be feasible, because only the
genome of virus will enter into the cell to function. Thus, it would be difficult for endogenous protein to
interact with it. With further brainstorming, we realised that there was a number of miRNAs existing in the cytoplasm,
which also showed discrepancy in expression between the normal and the tumor cell.  **With further study we found that the miRNA could bind to
its complementary sequence in certain parts, which had the potential to control the level of protein expression.**

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/ee/T--ZJU-China--Engneering-Pic3.png)

## **Design 2:** *How could we exploit it?*

We hoped that our products would only grow 'healthily' in cancer cells while in normal cells, they were incapable to proliferate, so we tried to use the difference of miRNA expression in the cytoplasm to control it. From a database, we chose some differently expressed miRNAs:**199a 195 22** and inserted their complementary sequences into the 3' UTR of the virus *E1A* gene, which is essential for the survival and multiplication of the virus. This design allowed the virus to act differently in these two types of cells, and that was how Viruguard played the role of "Viru" and "Guard" specifically.

![](https://static.igem.org/mediawiki/2021/0/0b/T--ZJU-China--Engneering_pic4.png)

**Fig.3** The different expression level of specific types of miRNA in normal and tumor cell lines.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/e7/T--ZJU-China--Engneering-Pic4.png)

## **Build 2:** *How can we build it?*

We designed the suicide switch by inserting the complementary sequence of **miRNA 199a,195 and 22** into the 3' UTR of the virus *E1A* gene. This design gave normal cells a weapon to escape the possible virulence from our product. E1A is an essential gene for virus proliferation. The combination between the free miRNAs and their complementary sequence in 3'UTR of the E1A would lead to its silence. And meanwhile, in tumor cells, due to the lower expression of those miRNAs, little effect will be put to viral viability and spread.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/7/7d/T--ZJU-China--Engneering-Cycle5.png)

## **Test 2:** *Do we get what we want?*

In order to examine the function of E1A-3'UTR, we transfected the 3UTR-eGFP plasmid vector into different cells to see whether eGFP expression level had coincident linear change with fluctuation of selected miRNA contents. The data showed a positive result, which indicated the feasibility and validation of our design.

#### [**BBa\_K3730013**](http://parts.igem.org/Part%3ABBa_K3730013)

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/5/56/T--ZJU-China--Engneering-Cycle6.png)

## **Learn 2:** *Could we further improve our specificity design?*

We had learned that those lower expressed miRNAs in tumor cells could be used to increase the expression level of E1A. But it could always be better to boost the gap of E1A content in normal cells and cancer cells. Therefore, to eliminate the miniature inhibitory effect of free miRNAs on the oncolytic virus in cancer cells, we designed a special sequence called 'miRNA sponge' under the control of tumor-specific promoter PhTERT, which was composed of a complementary tandem sequence of miRNAs 199a, 195, and 22. The mRNA transcribed from sponge DNA can absorb free miRNAs mentioned above through complementary binding, which competitively snatched the free miRNAs that were originally supposed to bind to 3'UTR of E1A. And simultaneously, in normal cells, because of the effect of phTERT, the miRNA sponge has little expression, thus E1A expression will still be inhibited totally.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/8/8c/T--ZJU-China--Engneering-Cycle7.png)

## **Improve 2:** *Is it enough for our design?*

**'miRNA sponge'** is composed of several complementary tandem repeats of each miRNA flanking by two restriction enzyme sites in our vector and could act as a molecule sponge to
absorb the miRNA in the cytoplasma. The secondary structure and miRNA binding affinity of sponge were evaluated by RNA fold web server and miRNAsong to make sure the miRNA binding sites were exposed and it had strong attraction to free miRNA.
As the level of the miRNA in cancer cells is relatively low, this mechanism would largely increase the treating effect of Viruguard in HCC cells.
Besides, we used tumor specific promoter *survivin* for the expression of the sponge to
avoid the incidence of insecurity in normal cells.

#### [**BBa\_K3730012**](http://parts.igem.org/Part%3ABBa_K3730012)

![](https://static.igem.org/mediawiki/2021/5/5c/T--ZJU-China--Engneering_Pic5.png)

**Fig.4** The construction of our miRNA sponge. The structure prediction by online tools.

Neverthelessly, with the design of killing and specificity, the virulence control measures were still more like 'remedial measures' than 'preventive measures' because they all focused on the virulence restriction after the virus had entered the cells. **Then it occurred to us that we could directly increase the specificity of the infection.**  We continued to improve our product.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/0/07/T--ZJU-China--Engneering-Circle1.png)

## **Research 3:** *How to improve virus recognition ability to tumor cells*

Now, our virus had a powerful weapon to kill liver cancer cells and an insurance to ensure that the virus could only replicate specifically in liver cancer cells which brought Viruguard to a new level. However, with further research conducted, we found that a better preventive measure to increase our safety. Let's start with how
adenoviruses invade cells.

Adenovirus infects cells by two successive interactions with cells. Firstly, Ad fiber protein
mediates its attachment to cells through the interaction with 46 kDa cell receptor CAR
(coxsackievirus and Ad receptor). Then, the adenovirus penton base protein and the integrin αvβ
3 and αvβ 5 binds to promote virus internalization. That is to say, the infection efficiency of Ad
infecting cells mainly depends on the expression level of CARs on the surface of target cells!

Surprisingly, most of cancer cells have low expression of CAR, and the expression of it is also
significantly reduced in hepatocellular carcinoma. This may be one of the reasons that current adenovirus
cancer therapy was not effective enough.

Many attempts have been made to solve the problem of low infection efficiency of virus to cancer
cells. However, these methods generally focus on improving the efficiency of infection which may somehow ignore the
specificity of infection. In this regard, we proposed a novel design that took both aspects into account.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/5/59/T--ZJU-China--Engneering-Circle2.png)

## **Imagine 3:** *What can we use to reach the goal?*

**Our initial idea was to target Glypican-3 (GPC3, as a universal membrane marker of liver cancer
cells)**, so that the virus can specifically recognize the protein and bind to liver cancer
cells. When it comes to specificity, it is natural think of antibodies. In this way, single chain variable
fragment (scFv) becomes a good choice. It is featured by low immunogenicity, high specificity and low
molecular weight which ensured the minimum impact would be put on the viral protein structure when constructing the fusion protein. Therefore, we **fused the fiber protein of adenovirus with the scFv of GPC3**, so that adenovirus can target HCC cells and then enter the cells through secondary interaction

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/ee/T--ZJU-China--Engneering-Pic3.png)

## **Design 3:** *How we use it?*

Mouse derived monoclonal antibodies GC33 were humanized by using complementary determinant region transplantation (CDR grafting). Using humanized GC33 can make it avoid being
recognized by human immune system in the early stage. The VH and VL sequences are as follows (blue is CDR region):

#### hGC33VHa:

QVQLVESGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARFYSYTYWGQGTLVTVSS

#### hGC33VLa:

DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK

![](https://static.igem.org/mediawiki/2021/7/77/T--ZJU-China--new-f4.png)

**Fig.5** The fusion protein combining the GPC3 receptor specifically, and reducing possible virulence to normal cells.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/e7/T--ZJU-China--Engneering-Pic4.png)

## **Build 3:** *How we build it?*

In order to prove that our fusion protein has little effect on the tertiary structure of the virus
fiber protein, so that it will not affect the assembly of the virus and further affect the virus titer,
**we used [predicted](https://zhanglab.ccmb.med.u            mich.edu/I-TASSER/) the three-dimensional structure of the fusion protein**. The results showed that it had little effect on the structure of knob of fiber.

![](https://static.igem.org/mediawiki/2021/b/b3/T--ZJU-China--Engneering_Pic7.png)

**Fig.6** The original structure of knob protein (left) and the structure prediction of fusion protein (right) . Red, the N-terminal of knob; Green, knob.

Before fusion of scFv and fibre protein, we were supposed to verify the activity and function of our scFv. Therefore, we chose prokaryotic expression to express scfv, and since *Escherichia coli* was the most common choice, it was selected for following experiments. We conducted a codon optimization for the amino acid sequence of scFv to ensure that it is suitable for expression in *E. coli*, and the gene sequence was synthesized by GenScript. We directly purchased fusion gene of scfv and fiber protein from Genscript as well.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/7/7d/T--ZJU-China--Engneering-Cycle5.png)

## **Test 3:** *Do we get what we want?*

We expected scFv to be expressed in a soluble form in E. coli, which is considered to be functional.**The results of Western-blot showed that scFv was successfully expressed and had considerable soluble expression.** Next, we verified the activity of scFv through a series of experiments, including Western-blot, ELISA, immunofluorescence and so on. The results met our expectations.

#### [**BBa\_K3730035**](http://parts.igem.org/Part%3ABBa_K3730035)

![](https://static.igem.org/mediawiki/2021/e/e6/T--ZJU-China--Engneering_Pic8.png)

**Fig.7** The exploration of the best condition for scFv expression.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/5/56/T--ZJU-China--Engneering-Cycle6.png)

## **Learn 3:** *What we did not know from our design?*

**In protein expression experiments, we managed to avoid rare codons in the protein, but failed,
so we chose Rosetta competent cells.** We will further select appropriate vectors, which contain appropriate
antibiotic type, promoter type, fusion label, and etc, to make our plasmid better match competent cells (possibly with
resistance), and have appropriate expression amount, soluble expression or inclusion body expression, and
purification method. And optimal conditions for expression were also to be discovered, such as induction
temperature, concentration, time, etc.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/8/8c/T--ZJU-China--Engneering-Cycle7.png)

## **Improve 3:** *Is it enough for our design?*

Although scFv did not affect the structure of adenovirus fiber protein in the tertiary structure prediction, further experiments were needed to confirm our prediction. **In the design of this part, what we were most worried about was not the off-target effect caused by the specificity of the antibody, but the effect of modification of the virus capsid protein put on the vitality of the virus itself, which was very important to ensure that the virus could have an efficient killing effect.**

Up to now, we have designed several parts to guarantee the safety of our product. However, we noticed that the current oncolytic virus treatment on the market always had the **problem of early immunoclearance by the immune system**, which could lead to compromising of therapeutic effect. Therefore, we had to find a way to temporarily allow our virus to escape from the clearance.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/0/07/T--ZJU-China--Engneering-Circle1.png)

## **Research 4:** *The immunoreaction to the virus*

We researched immunoreaction mechanism to adenovirus specifically in human body. **We learned that CpG is an episode of
DNA sequence in the virus genome, which could be recognized by the immune cell surface receptor
TLR9.** Once they bind to each other, TLR9 will switch into active form and trigger the downstream
immunoreaction. If we were able to block this interaction, the immune cell would not eliminate
Viruguard any more.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/5/59/T--ZJU-China--Engneering-Circle2.png)

## **Imagine 4:** *What can we use to reach the goal?*

In order to reach our goal of compromising immune resisitance, we had to find a competitive
sequence to wrest the TLR9. As the traditional recognition method is based on DNA sequence
complementary pairing, **the most feasible way is to find a sequence which could competitively
combine with TLR9.**

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/ee/T--ZJU-China--Engneering-Pic3.png)

## **Design 4:** *How we use it?*

**We got the TLR9i sequence from the manuscript published before.** This special sequence acted in the
same way as CpG sequence, which had higher affinity to the TLR9 receptor but it would not trigger the
following downstream cascades of immune reactions. Besides, as the DNA-receptor combination is temporary and they will disassociate when the virus fully gets into the cell, the silencing of immune system will not affect the normal immunal function.

![](https://static.igem.org/mediawiki/2021/5/54/T--ZJU-China--Engneering-pic8.png)

**Fig.8** TLR9i sequence will competitively cobine the GPC3 receptor, allowing temporary immunocamouflage.

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/e/e7/T--ZJU-China--Engneering-Pic4.png)

## **Build 4:** *How we build it?*

In order to verify the function of TLR9i, we first constructed ODN2216+TLR9i and ODN2216+Ctrl plasmids.

CpG ODNs are synthetic oligonucleotides that contain unmethylated CpG dinucleotides in specific
sequence contexts (CpG motifs). These CpG motifs could activate Toll-like receptor 9 (TLR9),
leading to strong immunostimulatory effects. ODN 2216 is a class A CpG ODN and is a ligand of
choice for human TLR9. We inserted ODN 2216 sequence into the plasmid, which could induce
high IFN-α production from peripheral blood mononuclear cell (PBMC)

**The following TLR9i sequence has a stronger affinity to TLR9, which prevents the activation of TLR9
and reduces the synthesis of IFN-α.** In order to enhance the inhibitory effect of TLR9i, we insert four
copies separated by short AAAAA linkers. ODN 2243, also known as ODN 2216 Control, is
designed as a negative control for the TLR9 agonist ODN 2216.

In the adenovirus genome, we chose to insert an oligonucleotide consisting of four copies of TLR9i separated by spacer into the 3′ UTR.

#### ODN2216 sequence

5’-ggGGGACGATCGTCgggggg-3’ (20 mer).

#### ODN 2243 (ODN 2216 Control) sequence

5’- ggGGGAGCATGCTGgggggg -3’ (20 mer)

#### TLR9i sequence:

5’- ttagggttagggttagggttaggg -3’ (24 mer)

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Engneering-arrow.png)

![](https://static.igem.org/mediawiki/2021/7/7d/T--ZJU-China--Engneering-Cycle5.png)

## **Test 4:** *Do we get what we want?*

We transfected ODN2216+TLR9i, ODN2216+ODN2243 and empty plasmid into PBMC respectively.
The supernatants were collected 24hrs and 48hrs after transfection and the levels of IFN-α produced in supernatants
were measured using a sandwich-type enzyme-linked immunosorbent assay
(ELISA) kit (Dakewe Biotech Co., Ltd.), according to the manufacturer’s instructions.

We found that PBMC transfected with ODN2216+TLR9i produced IFN- α significantly less than those transfected with ODN2216+OND 2243, which well verified the function of TLR9i. At the same time, PBMC transfected with empty plasmid produced least IFN- α among all other groups.

#### [**BBa\_K3730007**](http://parts.igem.org/Part%3ABBa_K3730007)

## References

[1] Gierach, I., Li, J., Wu, W. Y., Grover, G. J., & Wood, D. W. (2012). Bacterial biosensors for screening isoform-selective ligands for human thyroid receptors α-1 and β-1. FEBS open bio, 2, 247–253. https://doi.org/10.1016/j.fob.2012.08.002

[2] Gangopadhyay, J. P., Jiang, S., & Paulus, H. (2003). An in Vitro Screening System for Protein Splicing Inhibitors Based on Green Fluorescent Protein as an Indicator. Analytical Chemistry, 75(10), 2456–2462. DOI:10.1021/ac020756b

[3] Costa, S., Almeida, A., Castro, A., Domingues, L. (2014). Fusion tags for protein solubility, purification, and immunogenicity in Escherichia coli: the novel Fh8 system. Front. Microbiol., 5, 63, https://doi.org/10.3389/fmicb.2014.00063

[4] Costa, S. J., Almeida, A., Castro, A., Domingues, L., Besir, H. (2013). The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology. Appl Microbiol Biotechnol, 97, 6779–6791. DOI 10.1007/s00253-012-4559-1

[5] Plasmids 101: Origin of Replication. Retrieved July 25, 2020, from [https://blog.addgene.org/plasmid-101-origin-of-replication]

[6] Gonzalez-Flo, E., Alaball, M. E., Macia, J. (2020). Two-Component Biosensors: Unveiling the Mechanisms of Predictable Tunability. ACS Synthetic Biology, 9(6), 1328-1335. DOI: 10.1021/acssynbio.0c00010

[7] UniProtKB - P10828 (THB\_HUMAN). Retrieved October 22, 2020, from [https://www.uniprot.org/uniprot/P10828]

[8] Badran, A., Liu, D. (2015). Development of potent in vivo mutagenesis plasmids with broad mutational spectra. Nat Commun, 6, 8425. https://doi.org/10.1038/ncomms9425

[9] Chan, Y. K. , Wang, S. K. , Chu, C. J. , Copland, D. A. , & Church, G. M. . (2021). Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Science Translational Medicine, 13(580), eabd3438.

[10] Mortezagholi, S. , Babaloo, Z. , Rahimzadeh, P. , Namdari, H. , Ghaedi, M. , & Gharibdoost, F. , et al. (2017). Evaluation of tlr9 expression on pbmcs and cpg odn-tlr9 ligation on ifn-α production in sle patients. Journal of Immunopharmacology, 39(1), 11-18.

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Experiments

* [Immunocamouflage](#Immunocamouflage)
* [Targeted infection](#Targeted_infection)
* [Specific replication](#Specificreplication)
* [Oncolytic Virulence](#OncolyticVirulence)
* [Recombinant adenovirus experiment](#Recombinantadenovirusexperiment)
* [General Experiment](#GeneralExperiment)

Our experiments are composed of four main parts: **specifc recognition,miRNA controlled modulation, oncolytic virulence, and immunocamouflage.** We tried to prove the feasibility of the whole design. Besides the final construction of the oncolytic virus, we still need to build multiple vectors for individual part and mechanism certification. All the proof methods are widely used and reliable experiments.

# Immunocamouflage

#### Part 1 Construction of ODN 2216+Control, ODN 2216+TLR9i plasmid

From the InvivoGen, we got the sequences of ODN-2216、ODN-2243(Ctrl) and TLR9i(ODN-TTAGGG).

ODN 2216 is an A-class CpG ODN with a preference for human TLR9, which is characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3’ poly-G string. They induce high IFN-α production from peripheral blood mononuclear cells (PBMC). ODN 2243 (also known as ODN 2216 Control) is designed as a negative control for the TLR9 agonist ODN 2216.

ODN TTAGGG contains 4 repeats of the immunosuppressive TTAGGG motif which could inhibit immune activation by CpG-containing ODNs.

We entrusted the production of our plasmids, including plasmid ODN2216+Ctrl and plasmid ODN2216+TLR9i, to GenScript company, using the traditional restriction digestion and ligation method. For making inhibit the effect of TLR9i more obvious, we inserted four copies of TLR9i (ODN-TTAGGG) separated by short AAAAA linkers. We chose pcDNA3.1(+)-C-eGFP as our plasmid backbone, considering that this backbone has been used in our previously synthesized plasmids which could be conveniently taken as our control plasmid. Common restriction enzyme cutting sites EcoRI and BamHI were chosen in the meantime.

| Name | Sequence |
| --- | --- |
| ODN-2216 | 5'-ggGGGACGATCGTCgggggg-3' |
| ODN-2243 | 5'-ggGGGAGCATGCTGgggggg-3' |
| ODN-TTAGGG | 5'- ttagggttagggttagggttaggg-3' |
| ODN2216+TLR9i | 5'-ggGGGACGATCGTCggggggTTAGGGTTAGGGTTAGGGTTAGGGaaaaaTTAGGGTTAGGGTTAGGGTTAGGGaaaaaTTAGGGTTAGGGTTAGGGTTAGGGaaaaaTTAGGGTTAGGGTTAGGGTTAGGG-3' |
| ODN2216+Ctrl | 5'-ggGGGACGATCGTCggggggGGGGGAGCATGCTGGGGAGCATGCTGGGGAGCATGCTGGGGAGCATGCTGGGGGGGG-3' |

**Table 1-1.** Oligonucleotides sequences used in this study.

#### Part 2 Transfection of plasmids to PBMCs

PBMCs (Otwo Biotech Co., Ltd.) were plated in 24-well plates in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% Penestrep and maintained at 5% CO2 in air incubator at 37°C. PBMCs were transfected with the blank plasmid, ODN 2216+Ctrl, and ODN2216+TLR9i plasmids respectively. The cells were incubated for 48h. The collected supernatants were stored at 20°C until further use.

#### Part 3 Measurement of IFN-α

The supernatants were collected 48h after transfection and the levels of IFN-α production were measured in supernatants using a sandwich-type enzyme-linked immunosorbent assay (ELISA) kit (Dakewe Biotech Co., Ltd.), according to the manufacturer’s instructions.

# Targeted infection

#### Part 1 Dual plasmid transformation

The obtained scfv-pet28a (+) plasmid and pRARE plasmid from Rosetta strain is added to shuffle T7 competent cells in the equimolecular amount; 30 min on ice; 42℃ 90 s; 2 min on ice; added non-resistant LB medium for resuscitation for 1 h, and then coated with Kan+Cm+ double-antibody LB plate. The single colony obtained is the transformed strain.

#### Part 2 Expression and Detection of scFv protein

1. Shuffle T7 strain containing scfv-pET28a (+) and pRARE plasmids (hereinafter referred to as RS strain) is selected and cultured in Kan+Cm+ LB liquid medium overnight. Shuffle T7 strain and Rosetta strain (hereinafter referred to as S strain and R strain respectively) transferred into scfv-pET28a (+) plasmid are selected and cultured in Kan+ LB liquid medium and Kan+Cm+ LB liquid medium overnight.

2. The next day, according to the transfer ratio of 1:100, transfer the overnight cultured RS, S and R bacteria into fresh LB liquid medium containing antibiotics, shake for about 3-4h to the absorbance of 0.4-0.6.

3. Inductor IPTG is added for induction. Pay attention to setting the control group without IPTG.

**EXP A:** Strains R, S and RS: Temperature-37 ℃; IPTG concentration-1 mm; induction time-12 h

**EXP B:** RS strain - Temperature gradient (IPTG: 1mm, induction time 12h): 10℃, 20℃, 30℃, 37℃.

**IPTG concentration (temperature: 20℃, induction time: 12h)**: 0.1mm, 0.2mm, 0.5mm, 1mm. Induction time (temperature: 20 ℃, IPTG: 0.1mm): 6h, 12h, 18h, 24h.

4. The induced bacterial solution is centrifuged at 4000rpm at 4℃ for 3min; then resuspended the sediment using PBS, centrifuged and ished for 3 times.

5. Add an appropriate amount of (Xml+75ul) PBS for resuspension (at this time, protease inhibitor PMSF and lysozyme can be added), and absorb 75ul bacterial solution for retention.

6. Ultrasonic crushing until the bacterial solution is clear.

7. Centrifuge the crushed bacterial solution at 13000r at 4℃ for 10min, suck the supernatant (if you are doing a small amount of culture, only 75ul is needed, and the rest are discarded; if a large amount of culture needs using for protein purification, all are sucked out and transferred to a clean container); add XmL+75ul 8M urea solution to dissolve the sediment, and then suck 75ul.

8. Add 25ul 4×Loading buffer to the three tubes of 75ul sample in steps (5) and (7); boil at 100℃ for 10min (note that heavy objects can be pressed above the pipe to prevent the cover from exploding).

Store the prepared whole bacteria, soluble and inclusion body protein samples at -20℃ or directly use them for SDS-PAGE or Western blot experiments (see general protocol for experimental steps).

#### Part 3 Purification of soluble scFv protein

We expressed scFv with 6×Histidine tag in prokaryotic , so Ni-column affinity chromatography is used for purification.

1. Mix the Ni-Agarose Resin and add it to the chromatographic column and stand for 10 minutes at room temperature until the gel is separated from the solution. Open the liquid outlet at the bottom and let the ethanol flow out slowly by gravity.

2. Add 5 times the column volume of deionized water to the packed column, ish the ethanol, and then balance the column with 10 times the column volume of soluble binding buffer (10mm imidazole, 20mm Tris HCl, pH = 7.9, 0.5m NaCl). The sample can be loaded after balancing.

3. After the bacteria are collected, the bacteria are lysed by ultrasound until the bacterial solution is clarified. (note that protease inhibitors need to be added and the ultrasonic process is always in ice bath. Be sure to prevent temperature rise sharply or a large number of bubbles from causing denaturation and inactivation of soluble protein)

4. The soluble protein in the supernatant is collected by centrifugation at 13000 rpm and 4℃ for 10 minutes.

5. The bacterial lysate is diluted equally with soluble binding buffer, loaded on the column, the flow rate is 10 times the column volume/hour, and the flow-through solution is collected.

6. Rinse the column with a solid binding buffer 15 times the column volume to ish away the miscellaneous proteins.

7. Elute with an appropriate amount of solution solution buffer, and collect 1ml per tube.

8. After elution, the column is finished with deionized water 10 times the column volume, and then balanced with 20% ethanol 3 times the column volume. (the filler shall be immersed in ethanol) and stored at 2-8℃ after column sealing)

9. Take an appropriate amount of each sample in the purification process, add protein loading buffer and boil it at 100 ℃ for 10min for subsequent SDS-PAGE and Coomassie brilliant blue staining to analyze the purification effect.

10. Select scFv protein with high purity and concentration (if necessary, desalting and concentrating can be carried out with dialysis bag or ultrafiltration tube. See their instructions for the method); add 100% sterile glycerol of equal volume, and store it at - 20℃.

#### Part 4 Protein concentration determination

Considering that our protein sample is not very pure, we did not choose BCA protein concentration quantitative method to determine the concentration of scFv in the purified sample, but SDS-PAGE and Coomassie brilliant blue staining method.

1. Prepare standard concentration protein samples and purified scFv samples to be measured: dilute His-HRP protein with a purity of 90% of 0.8mg/ml into samples with his HRP concentrations of 400ug / ml, 200ug / ml, 100ug / ml, 50ug / ml, 25ug / ml and 12.5ug/ml, and prepare scFv purified samples. Add the protein loading buffer into the above samples and boil at 100℃ for 10min.

2. The above samples are stained with Coomassie brilliant blue after running SDS-PAGE.

3. Scan and take photograph.

4. Use Image J software to analyze the grayscale of the strip, make a standard curve, and calculate the scFv concentration in the purified sample.

#### Part 5 The activity of scFv is analyzed by Western blot

1. Detection of recombinant human GPC3 (rHGPC3) antigen by Western blot: firstly, prepare the protein sample of rHGPC3, add the protein loading buffer and boil at 100 ℃ for 10 min. Run SDS-PAGE(60V, 80V); conduct transmembrane (300mA, 1H); block with 5% BSA for 1h (Note: do not use skimmed milk powder, our experiment shows that scFv may bind to a protein in skimmed milk powder, resulting in negative results); purified scFv protein samples are diluted 200 times with 5% BSA and incubated with PVDF membrane at 4℃ overnight; ish for 3 times with TBST times; anti-His antibody is incubated at room temperature for 3h; ish for 3 times with TBST times; HRP conjugated secondary antibody is incubated at room temperature for 1h, and ish for 3 times with TBST; exposure and take photograph.

2. Detection of GPC3 antigen in HepG2 cells by a Western blot: first, prepare HepG2 cell lysate samples; When HepG cells in the six-well plate are full, 200 ul Cell lysis buffer is added; dissociate at a speed of 200rpm for 1h on a 4℃shaker; Add protein loading buffer and boil at 100℃ for 10min. Run SDS-PAGE(60V, 80V); conduct transmembrane (300mA, 1H); block with 5% BSA for 1h (Note: do not use skimmed milk powder, our experiment shows that scFv may bind to a protein in skimmed milk powder, resulting in negative results); purified scFv protein samples are diluted 200 times with 5% BSA and incubated with PVDF membrane at 4℃ overnight; ish for 3 times with TBST times; anti-His antibody is incubated at room temperature for 3h; ish for 3 times with TBST times; HRP conjugated secondary antibody is incubated at room temperature for 1h, and ish for 3 times with TBST; exposure and take photograph.

#### Part 6 Kd value of antibody is determined by enzyme-linked immunosorbent assay

1. HepG cells are inoculated into 24 well plates. Wait until the cell confluence reaches 100%.

2. Suck off the medium and ish once with 200ul PBS.

3. Add 200ul 4% paraformaldehyde to fix cells for 5min

4. Wash once with 200 ul PBS

5. For anti-GPC3 polyclonal antibody or scFv, add 1% BSA (prepared with PBS) and block at room temperature for 1h.

6. For scFv, scFv diluted to different concentrations with 1% BSA (prepared with PBS) is added and incubated at 4℃s overnight. ish for 3 times with PBS.

7. For polyclonal antibodies, PaAb diluted to different concentrations with 1% BSA (prepared with PBS) is added and incubated at room temperature for 3h. ish for 3 times with PBS.

8. For TLS11a, 5'-biotin labeled TLS11a of different concentrations prepared with PBS is added and incubated at room temperature for 2h. ish for 3 times with PBS.

9. For scFv, an anti-His antibody diluted by 1/1000 with 1% BSA is added and incubated at room temperature for 3h. ish for 3 times with PBS. Then add HRP-conjugated secondary antibody diluted with 1% BSA and incubate at room temperature for 1h. ish for 3 times with PBS.

10. For polyclonal antibody, HRP-conjugated secondary antibody diluted with 1% BSA is added and incubated at room temperature for 1h. ish for 3 times with PBS.

11. For TLS11a, streptavidin-HRP prepared by PBS is added and incubated at room temperature for 1h. ish for 3 times with PBS.

12. Add 200ul TMB chromogenic solution into each well and react at 37℃ for 15min. 200ul 2M HCl is added to terminate the reaction. The absorbance of 450 nm is measured by enzyme labeling instrument.

#### Part 7 Cellular immunofluorescence

1. Drop 10ul DMEM medium into 24 well plates, add special cell climbing sheet treated by TC, and inoculate HepG2 cells with a relatively low density.

2. After 48 hours, the cells adhered firmly to the sheet. Suck off the medium and ish it once with 200ul PBS.

3. Add 200ul 4% paraformaldehyde to fix for 5min.

4. Wash once with 200 ul PBS

5. For scFv, add 1% BSA (prepared with PBS) and block at room temperature for 1h. scFv diluted by 1/200 with 1% BSA is added and incubated at 4℃ overnight. ish for 3 times with PBS. Then add 1/1000 anti-His antibody diluted with 1% BSA and incubate at room temperature for 3h. ish for 3 times with PBS. Then FITC-conjugated secondary antibody diluted with 1% BSA is added and incubated in the dark at room temperature for 1h. Then dye with DAPI away from light for 2min and finished with PBS for 3 times. Take out the climbing slide, put it upside down on the glass slide dripping 3ul glycerol, observe and take photos with a confocal microscope.

6. For anti-GPC3 polyclonal antibody, 1% BSA (prepared with PBS) is added and blocked at room temperature for 1h. Polyclonal antibody diluted 1/1000 with 1% BSA is added and incubated at room temperature for 3h. ish for 3 times with PBS. Cy5-conjugated secondary antibody diluted with 1% BSA is added and incubated at room temperature in the dark for 1h. Then dye with DAPI away from light for 2min and ished with PBS for 3 times. Take out the climbing slide, put it upside down on the glass slide dripping 3ul glycerol, observe and take photos with a confocal microscope.

7. For TLS11a, 5'-Rox labeled TLS11a prepared with PBS is added and incubated at room temperature in the dark for 2h. ish for 3 times with PBS. Then dye with DAPI away from light for 2min and ished with PBS for 3 times. Take out the climbing slide, put it upside down on the glass slide dripping 3ul glycerol, observe and take photos with confocal microscope.

#### Part 8 Flow cytometry

1. When hepG2 cells of two 10cm dishes are full, they are digested with trypsin. Then centrifuge to collect all cells.

2. Wash twice with PBS, add 1.2ml PBS to suspension cells, divide them into 6 parts and add them into 6 EP tubes (each tube is 200ul).

3. 200ul of His-HRP (blank group) or His-scFv of 1.25ug/ml, 2.5ug/ml and 5ug/ml are added respectively and incubated at room temperature for 1h.

4. Washed with PBS for 3 times.

5. Add anti-His antibody(1/1000) and incubated at room temperature for 1h.

6. Washed with PBS for 3 times.

7. Add diluted FITC-conjugated secondary antibody and incubate at room temperature in the dark for 30 min.

8. Washed with PBS for 3 times.

9. Fluorescence analysis by flow cytometry.

# Specific replication

#### Overview

The design of this part starts with the difference of miRNA between HCC cell (HEPG2 cell line) and normal cell (HEK293T cell line), and we designed specific virus gene with a corresponding target sequence in its 3’UTR. Based on the virus gene with special 3’UTR, we added the design of sponge to enlarge the difference of miRNA.

Therefore, we measured the difference of miRNA content in HepG2 cell line and HEK293T cell line, constructed eGFP-3’UTR and sponge plasmids, and tested the function of them with miRNA added or not.

#### Part 1 Design of E1A-3'UTR

To make the Viruguard to distinguish cancer cells from normal cells, we chose three different miRNAs which was said to have different content in HCC cells and normal cells as shown in Table. 3-1 and the detailed distribution of these miRNAs in differnt cell typeswere further confirmed by atlas of human miRNA in Fig. 3-1 [1-3].

| Name | Sequence |
| --- | --- |
| has-miR-22-5p | AGUUCUUCAGUGGCAAGCUUUA |
| hsa-miR-195-5p | UAGCAGCACAGAAAUAUUGGC |
| hsa-miR-199a-5p | CCCAGUGUUCAGACUACCUGUUC |

**Table 3-1** The miRNA chosen to be including in 3’UTR of E1A. These three kinds of miRNA were previously confirmed to have different content in HCC cells and normal cells.

![](https://static.igem.org/mediawiki/2021/f/fc/T--ZJU-China--nweb1.png)
![](https://static.igem.org/mediawiki/2021/b/b8/T--ZJU-China--nweb2.png)

**Fig. 3-1** The distribution of miRNA22 in normal human cells.

The miRNA195 and 199 were examined in the same way. We chose Hek293T cells as our comparison cell in later experiments because the kidney cells were predicted to have lower expression of these 3 kinds of miRNA compared with other normal cells.

After specific miRNAs were selected, how to insert each miRNA to 3’UTR of E1A became a problem. With the tool provided online, we obtained the reverse complementary of miRNA first and designed the spacer which separated each miRNA according to Saetrom’s study in Fig. 3-2, the binding affinity of miRNA to 3’UTR were also predicted [4,6].

![](https://static.igem.org/mediawiki/2021/5/57/T--ZJU-China--Experiment-expFig3-2.png)

**Fig. 3-2** The design of E1A-3’UTR. Each miRNA copy were seperated by specific spacers.

![](https://static.igem.org/mediawiki/2021/1/19/T--ZJU-China--Experiment-expFig3-3.png)

**Fig. 3-3** The affinity of each miRNA to its complementary sequence in E1A-3’UTR (named "sponge" in this figure). It indicated that miRNA were likely to bind to 3’UTR that we designed.

#### Part 2 Design of miRNA sponge

3'UTR-E1A should have been efficient enough to slience the E1A, but could we make the Viruguard safer by increasing the difference of miRNA content between normal cells and HCC cells. Thus we decided to design a miRNA sponge like Fig. 3-4 which constitutes of tandem repeats of all three kinds of miRNAs to absorb free miRNA under the control of tumor specific promoter according to Kluiver’s manuscript [5].

![](https://static.igem.org/mediawiki/2021/5/5a/T--ZJU-China--Experiment-expFig3-4.png)

**Fig. 3-4** The sequence of miRNA sponge.

The sponge is composed of several complementary tandem repeats of each miRNA flanking by two restriction enzyme sites.

The secondary structure and miRNA binding affinity of sponge were evaluated by RNA fold web server and miRNAsong to make sure the miRNA binding sites were exposed and it had strong attraction to free miRNA which was illustrated in Fig. 3-5 and Fig. 3-6 [5,6].

![](https://static.igem.org/mediawiki/2021/5/5f/T--ZJU-China--Experiment-expFig3-5.png)

**Fig. 3-5** miRNA sponge secondary structure prediction.

The green paired bases had relatively low binding affinity which would not affect the binding of miRNA while the red bases had tight binding. Both possible linear and circular structure were shown above.

![](https://static.igem.org/mediawiki/2021/b/b1/T--ZJU-China--Experiment-expFig3-6.png)
![](https://static.igem.org/mediawiki/2021/c/c7/T--ZJU-China--Experiment-expFig3-6-2.png)

**Fig. 3-6** The affinity and kinetics of binding of each miRNA to its complementary sequence in miRNA sponge.

It indicated that our miRNA sponge were likely to absorb the free miRNA in cytosol.

#### Part 3 Construction of 3’UTR, sponge, and null plasmid

p3UTR and pSp were generated by Genescript.

To eliminate the living burden put on cells by plasmid transfection, null plasmids for p3UTR and pSp were constructed by one step cloning kit for negative control.

Polymerase chain reaction was performed in PCR machine together with KOD high fidelity polymerase mixture including flanking primers (Null\_1R & Null\_1F) as shown in Fig. 3-7. The phosphate group was added to sticky ends of PCR product by catalysis of PNK enzyme which were then connected by T4 ligase overnight. The concentration of PCR products was measured by nanodrop. The successful construction of empty plasmid was confirmed by agarose electrophoresis with primers 3UTR-EMPTY-F and 3UTR-EMPTY-R in Fig. 3-7 as well. Null plasmids were then transformed to DH5α and inoculated to Amp(+) plate. After incubating at 37℃ overnight, single bacterial colony was picked and shaking in liquid LB medium for 6 hours. Colonial PCR was performed by Taq polymerase to further illustrate the correct construction of null plasmid, which was followed by second generation sequencing by genescript.

![](https://static.igem.org/mediawiki/2021/3/37/T--ZJU-China--Experiment-expFig3-7.png)

**Fig. 3-7** Plasmid map of 3UTR.

Primers Null\_1R and Null\_F are used to construct p3'Unull. Primers 3UTR-EMPTY-F and 3UTR-EMPTY-R are used to verify the successful construction of p3’Unull. Using p3UTR as an example here, the construction of pSp and pSp null are same with it.

#### Part 4 Construction of replenishment plasmid

shRNA 22,195, 199 short single chain fragments was purchased. Annealing was performed under the condition of gradient descending temperautre and the products was inserted to pRNAiU6 by digestion of speedy cut XhoI and HpaI under 37℃ for 30mins. PNK enzyme and T4 ligase were used for connection of ending nucleotide. The product (pRNAiU6.2-22, pRNAiU6.2-195, pRNAiU6.2-199a) was examined and processed in the same way with null plasmid.

#### Part 5 miRNAs content measurement

Firstly, we chose miR-199a, miR-195, miR-22 from miRNA which had higher expression in normal cells than HCC cells among miR-195, miR-199a, miR-200a, miR-125a, miR-191, miR-22 and etc [1,2].

Then we used Bulge-Loop primer set from Ribobio to detect miRNA expression and differential analysis based on qPCR detection technology, and we chose U6-1 as our internal reference which was a component of splicing complex. The design of our experiment was illustrated in Table. 3-2.

![](https://static.igem.org/mediawiki/2021/8/8e/T--ZJU-China--Experiment-expTable3-2.png)

**Table 3-2** The control design of our experiment.

#### Part 6 Transfection of selected plasmids to HepG2 and Hek293T

p3UTR, pSp and their empty plasmids were transformed to BL21Plasmids were extracted by endofree plasmid midi kit after shaking overnight. The concentration of plasmid extracted was measured by nanodrop.

We placed HepG2 and Hek293T cells per well in 100μL of complete growth medium 12-24 hours prior to transfection. At the time of transfection, the confluency of cells were 70%-90% to ensure the transfection efficacy. In order to examine the function of E1A-3'UTR, HepG2 and Hek293T were transfected with 3’UTR-pcDNA3.1-N-eGFP, 3’UTRempty-pcDNA3.1-N-eGFP (p3U and p3Unull, 3Unull also called 3Uempty or 3ue here). After 24h, we use the microplate reader to detect the intensity of fluorescence. Living cell count were determined by CCK8, the absorption of which at 450nm indicated the number of living cell. The fluorescence microscope was used to capture the cells at 4× and 10× (objective) for three dark visions and three bright ones each well.

We also designed plasmids called pRNAiU6.2-22, pRNAiU6.2-195, pRNAiU6.2-199a to increase the quantity of these miRNA in HepG2 as described previously. That was how we could know if the effect of 3UTR was actually caused by miRNA or other variables. Thus, we transfected 3u and one of these plasmids above into HepG2. The group called 3u+u6 was the controlled group, and u6 referred to pRNAiU6 without extra part.

As for sponges, the cells were also transfected with the sponge-pcDNA3.1-P2A-C and 3’UTR-pcDNA3.1-N-eGFP at the same time. To figure out the proper ratio of these two types of plasmids, we also tried to transfect different ratios of plasmid, the result of which indicated the optimized design of the final virus. The quantity sum of the plasmids transfected were controlled as the same. The processing of plate stayed the same with what we did in 3U transfection. Other details about the arrangements of transfection are shown in the following supplementary materials.

The exampled experiment were shown below in Fig. 3-8.

![](https://static.igem.org/mediawiki/2021/9/9c/T--ZJU-China--Experiment-expFig3-8.png)

**Fig. 3-8** 24well plate and 96 well plate used for transfection. This module aimed to test the function of 3’UTR and miRNA sponge.

We provide some [supplementary materials.](https://static.igem.org/mediawiki/2021/3/35/T--ZJU-China--maozhuo.pdf)

# Oncolytic Virulence

#### Part 1 shRNA expression plasmid

##### **(1) shRNA sequences acquisition**

With the help of thermofisher online shRNA design tools, BLOCK-iT™ RNAi Designer[8], we are going to find the key sequences in our targeting genes for RNAi.

![](https://static.igem.org/mediawiki/2021/8/8d/T--ZJU-China--Experiment-expFig4-1.png)
![](https://static.igem.org/mediawiki/2021/3/31/T--ZJU-China--Experiment-expFig4-1-2.png)

**Fig. 4-1** The top ten shRNA sequence designed for (a) hTERT and (b)VEGFA gene interference.

![](https://static.igem.org/mediawiki/2021/f/fb/T--ZJU-China--Experiment-expFig4-2.png)
![](https://static.igem.org/mediawiki/2021/d/df/T--ZJU-China--Experiment-expFig4-2-2.png)

**Fig. 4-2** Three types of siRNA which combine different area of the gene sequence had been chosen for interfering each target gene. By adding custom loop sequence we had gained the shRNA primers for structure construction. (a) hTERT targeting shRNA primers. (b) VEGFA targeting shRNA primers.

##### **(2) Plasmid construction**

**1. shRNA annealing**

After loading single-stranded shDNA into PCR tube, it will be heated to 95℃ then cooled by a gradient to 24℃ to get dsDNA. The temperature gradient: 37℃ 30min; 95℃ 5min; 95℃ 10s; 85℃ 30s; 75℃ 30s; 60℃ 15s; 55℃ 15s; 50℃ 15s; 45℃ 30s; 35℃ 15s; 24℃ 15s; 12℃ forever for storage temporarily.

**2. Plasmid linearization**

We use specific restriction endonuclease ( XhoI and HpaI ) to cut the plasmid so that it will have the same sticky ends as what we designed in shRNA. The detail of digestion experiment please see the supplementary file. Agarose gel electrophoresis is used to verify the success of linearization.

**3. Connection**

We use T4-ligase to link shDNA to the linearized plasmid. The reaction mixture will be incubated at 22℃ for ten minutes to achieve maximum bonding efficiency. Then the constructed plasmid will be transformed into DH5α bacteria. The transformed bacteria will be inoculated on kanamycin resistant medium. Then the growing colony will be used to carry on colony PCR and agarose gel electrophoresis to ensure the connection is successful. Finally we send the bacteria colony to the company for sequencing.

#### Part 2 Effects of shRNA on cancer cell survival related genes

The designed shRNA will be processed in the nucleic of the cell and then interfere with the targeting gene through the RNAi mechanism. We want to explore the efficiency on transcription silence of our designed shRNA sequence and see whether it will cause translation suppression and greater cell apoptosis. So we are going to verify the design from a different stage of the gene expression.

Firstly, we will use a direct way to see how our mechanism will influence the survival of the cell. HepG2 cells were planted in six-hole plate one day before transfection and after 24h or 48h, they are collected. By using the Annexin-V FITC/PI method, we stained the apoptosis cells into red and by checking the intensity of the fluorescence, we were able to calculate the relative dead rate of the cell.

Next, we decide to make sure shRNA is able to reduce the mRNA level of VEGFA and hTERT. HepG2 cells were planted in six-hole plate one day before transfection and after 24h or 48h, they are collected. Then we use the qRT-PCR to see after two days of transfection, how the transcription level will be influenced.

| Group | Time | Dose of shRNA plasmid |
| --- | --- | --- |
| Blank | 24,48h | 0 |
| GADPH | 24,48h | 1,2mg |
| Scramble | 24,48h | 1,2mg |
| hTERT-1,2,3(Three individual holes) | 24,48h | 1,2mg |
| VEGFA-1,2,3(Three individual holes) | 24,48h | 1,2mg |

**Table 4-1.** The design of our transfection experiment to find ,the best condition of detecting the effect of shRNA on transcription level. Cells are cultivated in 6 hole plate. There are around 2x10^5 cells in each hole.

Finally, we are going to see how the protein expression changes along with the transfection of shRNA plasmids. We collected the cell cultivated in six-hole plate and extracted their protein after one, two or three days of transfection and then conducted the Western Blotting to check the expression level of the protein.

| Group | Time | Dose of shRNA plasmid |
| --- | --- | --- |
| Blank | 24,48,72h | 0 |
| GADPH | 24,48,72h | 1,2mg |
| Scramble | 24,48,72h | 1,2mg |
| hTERT-1,2,3(Three individual holes) | 24,48,72h | 1,2mg |
| VEGFA-1,2,3(Three individual holes) | 24,48,72h | 1,2mg |

**Table 4-2.** The design of our transfection experiment to find the best condition of detecting the effect of shRNA on protein expression. Cells are cultivated in 6 hole plate. There are around 2x10^5 cells in each hole.

#### Part 3 Specific expression promoter

Next, we are going to examine whether the specific expression promoter work as our expectation. By transfecting the survivin-EGFP or htert-EGFP plasmid into cancer cell lines HepG2 and normal cell line, we decide to show the expression difference by calculating the green fluorescence intensity of different groups.

| Cell type | Time | Type of promoter |
| --- | --- | --- |
| Hek293T | 24,48h | *survivin,htert* |
| HepG2 | 24,48h | *survivin,htert* |

**Table 4-3.** The design of our transfection experiment to find the best condition of detecting the expression efficiency of the two types of promoters in different cell lines. Cells are cultivated in 6 hole plate. There are around 2x105 cells in each hole.

# Recombinant adenovirus experiment

After verifying that each part of the design meets our assumptions, we need to detect the actual effect of virus guard from the virus level, so we need to do some virus experiments. Here is our plan for virus experiment.

#### Part 1 Determination of virus infection and replication ability in normal hepatocyte Lo2 and hepatoma cell HepG2

We can characterize the infection and replication ability of the virus by measuring the titer or MOI of the virus to two cells.

**(1) Determination of virus titer**

a. On the day before infection, two six well plates (5 per well) were inserted×105 cells) were inoculated into HepG2 and LO2 cells, respectively.

b. 24 hours after inoculation, we counted HepG2 and LO2 cells in six wells using the blood cell counting plate method to determine the actual number of cells.

c. The remaining holes are used to inoculate the virus.

d. After 20 hours of infection, change to dnaasei fresh medium, digest the residual plasmid DNA at 37 ℃ for 15 minutes, and then change to 2ml normal medium for 48 hours.

e. DNA extraction, virus quantification.

**(2) Virus MOI assay**

a. The day before infection was inoculated, so that the cell confluence was 30-40% the next day.

b. Set infection groups with different MOI values, such as infection groups of 1, 2, 5, 10, 20, 50, 100, and make two holes respectively.

c. After 12-24 hours, discard the culture supernatant, remove the virus without infected cells, and replace with fresh culture medium.

d. After 3-4 days of infection, the cells can be observed under a fluorescence microscope. The cells will be infected and express fluorescence. The proportion of cell infection can be estimated visually by taking photos or calculated according to the number of pictures.

e. Genomic DNA can also be extracted from digested cells, and the proportion of integrated virus and cells can be obtained by quantitative PCR method introduced in titer determination. Note that if the infected animal cells need to use the internal reference genes of the corresponding species.

#### Part 2 Effect of virus infection on cell viability

The recombinant adenovirus infected LO2 normal hepatocytes and HepG2 hepatoma cells, respectively. Group settings are: A: LO2, B: LO2+virus, C: HepG2, D: HepG2+virus. Each group has three multiple holes. The cell viability was detected at 0h, 24h, 48h, 72h and 96h after infection. The detection method was CCK8 detection method, which was detected by flow cytometry or enzyme-labeling instrument.

#### Part 3 Determination of activation of immune response by virus

PBMC cells were infected with control adenovirus and recombinant adenovirus, with three replicates in each group. After 24 hours, the supernatants of the two groups were collected and the IFN-α in the supernatants was detected by ELISA.

# GENERAL EXPERIMENT

We provide some supplementary materials of general experiment. Please see this [File.](https://static.igem.org/mediawiki/2021/3/39/T--ZJU-China--LINHE.pdf)

## References

[1] Elhendawy, M., Abdul-Baki, E. A., Abd-Elsalam, S., Hagras, M. M., Zidan, A. A., Abdel-Naby, A. Y., ... & Abdou, S. (2020). MicroRNA signature in hepatocellular carcinoma patients: identification of potential markers. Molecular Biology Reports, 47(7), 4945-4953.

[2] Murakami, Y., Yasuda, T., Saigo, K., Urashima, T., Toyoda, H., Okanoue, T., & Shimotohno, K. (2006). Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene, 25(17), 2537-2545.

[3] De Rie, D., Abugessaisa, I., Alam, T., Arner, E., Arner, P., Ashoor, H., ... & De Hoon, M. J. (2017). An integrated expression atlas of miRNAs and their promoters in human and mouse. Nature biotechnology, 35(9), 872-878.

[4] Saetrom, P., Heale, B. S. E., Snøve, O., Aagaard, L., Alluin, J., & Rossi, J. J. (2007). Distance constraints between microRNA target sites dictate efficacy and cooperativity. Nucleic Acids Research, 35(7), 2333–2342.

[5] Kluiver, J., Slezak-Prochazka, I., Smigielska-Czepiel, K., Halsema, N., Kroesen, B. J., & van den Berg, A. (2012). Generation of miRNA sponge constructs. Methods, 58(2), 113-117.

[6] Barta, T., Peskova, L., & Hampl, A. (2016). miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico. Scientific reports, 6(1), 1-8.

[7] Gruber, A. R., Lorenz, R., Bernhart, S. H., Neuböck, R., & Hofacker, I. L. (2008). The vienna RNA websuite. Nucleic acids research, 36(suppl\_2), W70-W74.

[8] http://rnaidesigner.thermofisher.com/rnaiexpress

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Result

* [Immunocamouflage](#Immunocamouflage)
* [Targeted-infection](#Targeted-infection)
* [Specific replication](#Specific replication)
* [Oncolytic Virulence](#Oncolytic Virulence)
* [Virus experiments](#Virus experiments)
* [References](#References)

The results of all our design will be shown in this page. So far we had finished all our scheduled experiments and the results had proved the reliability and feasibility of our design to some extent. However, some picture may be not that beautiful because of the lack of time, and we are still conducting experiments for a better results. Besides, the results of how our designs work in the packaged oncolytic virus will soon be added.

# 1. Immunocamouflage

#### Part 1.1 Plasmid construction

From the InvivoGen[1], we got the sequences of ODN-2216、ODN-2243(Ctrl) and TLR9i(ODN-TTAGGG).

ODN 2216 is an A-class CpG ODN with a preference for human TLR9, which are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3' poly-G string. They induce high IFN-α production from peripheral blood mononuclear cells (PBMC). ODN 2243 (also known as ODN 2216 Control) is designed as a negative control for the TLR9 agonist ODN 2216.

ODN TTAGGG contains 4 repeats of the immunosuppressive TTAGGG motif which could inhibit immune activation by CpG-containing ODNs.

In order to verify the effect of TLR9i, we directly linked ODN2216 sequence with TLR9i sequence, inserted it into pcDNA3.1(+)-C-eGFP plasmid through traditional restriction digestion and ligation method and named it ODN2216+TLR9i. For making the inhibit effect of TLR9i more obvious, we inserted four copies of TLR9i (ODN-TTAGGG) separated by short AAAAA linkers. In the same way, we constructed a control plasmid named ODN2216+Ctrl.

| Name | Sequence |
| --- | --- |
| ODN-2216 | 5'-ggGGGACGATCGTCgggggg-3' |
| ODN-2243 | 5'-ggGGGAGCATGCTGgggggg-3' |
| ODN-TTAGGG | 5'- ttagggttagggttagggttaggg-3' |
| ODN2216+TLR9i | 5'-ggGGGACGATCGTCggggggTTAGGGTTAGGGTTAGGGTTAGGGaaaaaTTAGGGTTAGGGTTAGGGTTAGGGaaaaaTTAGGGTTAGGGTTAGGGTTAGGGaaaaaTTAGGGTTAGGGTTAGGGTTAGGG-3' |
| ODN2216+Ctrl | 5'-ggGGGACGATCGTCggggggGGGGGAGCATGCTGGGGAGCATGCTGGGGAGCATGCTGGGGAGCATGCTGGGGGGGG-3' |

**Table 1.** Oligonucleotides sequences used in this study.

![](https://static.igem.org/mediawiki/2021/e/ef/T--ZJU-China--ResultsP2Fig1.png)

**Figure. 1-1** Verification of our plasmid construction.
(a) Agarose electrophoresis of ODN2216+TLR9i before and after digestion. Lane 1 is ODN2216+TLR9i plasmid in three conformations. Lane 2 is ODN2216+TLR9i plasmid digested by EcoRI and MluI. Lane M is 1 KB ladder. (b) Agarose electrophoresis of ODN2216+Ctrl before and after digestion. Lane 1 is ODN2216+Ctrl plasmid in three conformations. Lane 2 is ODN2216+Ctrl plasmid digested by EcoRI and MluI. Lane M is 1 KB ladder. (c) 1 KB ladder. Each band are marked with corresponding length.

#### Part 1.2 IFN-α concentration following transfection

We transfected ODN2216+TLR9i, ODN2216+TLR9i and empty plasmid into PBMC respectively. The supernatants were collected 48h after transfection and the levels of IFN-α production were measured in supernatants using a sandwich-type enzyme-linked immunosorbent assay (ELISA) kit (Dakewe Biotech Co., Ltd.), according to the manufacturer's instructions.

![](https://static.igem.org/mediawiki/2021/0/0e/T--ZJU-China--ResultsP2Fig2.png)

**Figure. 1-2** ELISA plate detecting the level of IFN-α in standard samples and supernatants. A1-A8, B1-B8 are cytokine standards of IFN-α. The concentrations from one to eight are 500, 250, 125, 62.5, 31.25, 15.625 and 7.8 pg/mL. C1-C4 is ODN2216+Ctrl group. C5-C8 is ODN2216+TLR9i group. D1-D4 is blank group.

Due to the low transfection efficiency of traditional liposome transfection and electroporation transfection methods on immune/hematology related cells such as PBMC, ODN2216+TLR9i group, and the blank group did not reach the detection limit, but we can see the difference in absorbance. The absorbance value of ODN2216+Ctrl group was significantly higher than that of ODN2216+TLR9i group. Therefore, to an extent, it might suggest that TLR9i can compete to bind TLR9, triggering less downstream signal transduction, thus reducing the production of IFN-α and decreasing the immune response. As the virus transfection efficiency is higher, we will further explore in the follow-up virus experiment.

![](https://static.igem.org/mediawiki/2021/2/25/T--ZJU-China--ResultsP2Fig3.png)

**Figure. 1-3.** Absorbance at 450 nm after transfection of different plasmids. Data are shown as mean and standard difference. p < 0.001.

# 2. Targeted-infection part

#### Part 2.1 Our fusion strategy

**(1) Design of fusion protein**

Our aim is to fuse the single-chain variable antibody(scFv) of GPC3 to the C-terminal of virus coat protein, fiber. In order to achieve this, we used (G)4S flexible joint to connect the heavy chain variable region and light chain variable region of anti-GPC3 antibody, resulting in scFv of anti-GPC3. Then scFv is fused to the C-terminal of fiber protein through (G)4S flexible joint so that scFv will be expressed together with the fiber protein of the virus and present on the surface of the virus (as is shown in Figure 2-1).

![](https://static.igem.org/mediawiki/2021/f/ff/T--ZJU-China--model-1.png)

**Figure. 2-1** Strategy of fiber and scFv fusion.

**(2) Protein and DNA sequence of scFv**

The following figure shows the amino acid sequence of the fusion protein of fiber knob region (C-terminal) and scFv. The blue part shows the fiber protein knob region, and the red part shows VH and VL regions of anti-GPC3 antibody.

![](https://static.igem.org/mediawiki/2021/9/92/T--ZJU-China--Results-Pic5.png)

**Figure. 2-2** The amino acid sequence of fiber knob-scfv protein.

The following figure shows the DNA sequence of scFv. The red part is the VH and VL regions of anti-GPC3 antibody.

![](https://static.igem.org/mediawiki/2021/a/ac/T--ZJU-China--Results-Pic6.png)

**Figure. 2-3** The DNA sequence of scFv.

**(3) 3D structural prediction of fusion protein**

We know that the strategy of modifying the virus fiber protein may greatly affect the assembly of fiber trimer, further affect the efficiency of virus intracellular assembly and amplification, and affect the virus titer[2]. Therefore, we made a de novo prediction on the structure of the fusion protein by an online website[3]. Fig. 2-4 shows a three-dimensional structure of the knob region trimer of adenovirus type 5 fiber protein downloaded from the PDB structure database[4]. The results (Fig. 2-5) show that the fusion protein did not affect the structure of the fiber protein knob region.

![](https://static.igem.org/mediawiki/2021/2/20/T--ZJU-China--S1.jpeg)

**Figure. 2-4** 3D structure (trimer) of knob region of adenovirus type 5 fiber protein. (a) Bottom view, (b) side view. The spherical model shows the C terminal.

![](https://static.igem.org/mediawiki/2021/b/ba/T--ZJU-China--s2.jpeg)

**Figure. 2-5** Protein structure ab initio prediction (I-TASSER). (a) The prediction of 3D structure of fiber knob. Red shows the N terminal, green shows the main body of the fiber knob, and yellow shows the C terminal. (b) The prediction of 3D structure of fusion protein. Red shows the N terminal of the fiber knob, green shows the main body of the fiber knob, and yellow shows the C terminal of the fiber knob. Grey shows the flexible joint, purple shows the VH of scFv, and orange shows the VL of scFv.

#### Part 2.2 Prokaryotic expression and purification of anti-GPC3 scFv protein

**(1) Construction of prokaryotic expression plasmid of anti-GPC3 scFv protein**

Since the virus packaging takes a long time and the virus experiment has higher requirements for the laboratory, we decided to express scFv protein in prokaryotic first, and then verify its activity. The prokaryotic expression vector we selected is pET28a (+), which has a T7 promoter (controlled by lac operon) and kanamycin resistance. Then we constructed scFv into the target vector by enzyme digestion (EcoR I and Hind III restriction endonuclease) and T4 ligation. As is shown in Fig. 2-6, we successfully inserted the DNA sequence (801bp) of scFv into the target vector.

![](https://static.igem.org/mediawiki/2021/f/f9/T--ZJU-China--Results-Pic9.png)

**Figure. 2-6** Construction of scFv prokaryotic expression plasmid. Lane1: DNA ladder, lane2: pET28a(+) empty plasmid, lane3: pET28a(+) vector was digested by enzyme EcoR I and Hind III, lane4: scfv-pet28a(+) expression plasmid, lane5: scfv-pet28a(+) vector was digested by enzyme EcoR I and Hind III.

**(2) Solution to rare codon problem**

After the construction of scFv expression plasmid, we transformed it into shuffle T7 competent cells for protein expression, but it was found that scFv protein was not successfully expressed by SDS-PAGE. After checking our sequence, we found what the problem was. It turned out that although we optimized the codons of VH and VL regions of scFv, we ignored whether the flexible joints connecting them had rare codons. Unfortunately, it did. Therefore, we know that Rosetta competent cell contains a plasmid that can express rare codons, pRARE [5](as shown in Fig. 2-7). So this may help us express scFv successfully.

![](https://static.igem.org/mediawiki/2021/6/69/T--ZJU-China--Results-Pic10.png)

**Figure. 2-7** The map of pRARE helper plasmid.

**(3) Dual plasmid co-transformation strategy**

Since shuffle T7 strain constitutively expresses disulfide isomerase DsbC compared with Rosetta strain, this may help more soluble expression of foreign proteins. Therefore, we co-transformed scFv expression plasmid and pRARE helper plasmid into shuffle T7 strain. After dual antibiotics screening(Kan and Cm), we successfully obtained the strain carrying double plasmid (as shown in Fig. 2-8).

![](https://static.igem.org/mediawiki/2021/7/7d/T--ZJU-China--Results-Pic1.png)

**Figure. 2-8** ScFv-pET28a(+) expression plasmid and helper plasmid pRARE were co-transformed into shuffle T7 strain. Lane1: DNA ladder, lane2: pRARE helper plasmid, lane3: scfv-pET28a(+) expression plasmid, lane4: pRARE helper plasmid and scfv-pET28a(+) expression plasmid were co transformed into shuffle T7 strain, and then the plasmid was extracted.

**(4) Detection of Protein expression——selection of expression strain**

We compared the expression of scFv protein in several different hosts, and the results are shown in Fig. 2-9. Before IPTG induction, scFv protein was almost not expressed, indicating that our expression plasmid has strong robustness. After induction, scFv protein was successfully expressed. Compared with shuffle T7 strain carrying only expression plasmid, Rosetta strain carrying both pRARE plasmid and expression plasmid and shuffle T7 strain carrying both expression plasmid and pRARE plasmid had significantly enhanced protein expression. In shuffle T7 strain carrying only expression plasmid, the protein mainly existed in soluble form, but the amount was a little small. When two plasmids exist in the same host, the expression of total protein and soluble protein increased significantly, but many proteins also appeared in the inclusion body. However, only in Rosetta strain, the expression of soluble protein was very small. Our results also confirmed the rationality of the dual plasmid transformation strategy [6].

![](https://static.igem.org/mediawiki/2021/c/c4/T--ZJU-China--Results-Pic12.png)

**Figure. 2-9** Expression of scFv in different hosts. S: Shuffle T7 strain carrying expression plasmid, R: Rosetta strain carrying expression plasmid, RS: Shuffle T7 strain carrying both pRARE plasmid and expression plasmid. Bef.ind: before induction, Af.ind: after induction, Af.ind.sou: souble protein after induction, Af.ind.inc: inclusion protein after induction.

**(5) Exploration of optimal expression conditions**

Although we successfully expressed scFv protein, most of the protein still existed in the form of an inclusion body. We extracted and purified the inclusion body protein and verified that it had no activity. In addition, the refolding steps of inclusion body protein are very complex and can not guarantee 100% recovery of activity, so we chose soluble protein. Therefore, we explored the best conditions, including temperature, inducer concentration, and induction time, in order to obtain the most soluble expression.

Firstly, the protein can have time enough to be folded by reducing the speed of protein synthesis. Therefore, it can be achieved by reducing the temperature and the induced IPTG concentration. We selected four temperature gradients of 10 ℃, 20 ℃, 30 ℃ and 37 ℃. The results showed that with the decrease of temperature, the expression of total scFv protein decreased, but the expression of soluble protein first increased and then decreased (as shown in Fig10 a). We obtained that the optimum expression temperature for soluble protein was 20 ℃. That may be because there is higher dissolved oxygen at low temperature to prevent the anaerobic growth of bacteria from producing a large amount of acetic acid; Secondly, the half-life of antibiotics was longer and the plasmid stability was higher when cultured at low temperature. Moreover, protein has a lower degradation rate and soluble protein has higher stability at low temperatures. Finally, some researchers also believe that low temperature can change the dynamics of peptide folding and increase the amount of correctly folded proteins.

Then, we investigated the effects of different IPTG concentrations on soluble protein expression at 20 ℃. Results, as shown in Fig10 b, the expression of total protein, increased with the increase of IPTG concentration within the given four gradient ranges. However, the detection of soluble protein showed that there was no significant relationship between soluble protein expression and IPTG concentration. This shows that even at low IPTG concentration, the expression of total protein is still excessive, so there are still enough proteins to enter the correct folding process.

Finally, we explored whether the induction time would significantly affect the expression of soluble protein. Results, as shown in Fig10 c, the expression of total protein and soluble protein, increased with the increase of induction time, but the increase of protein expression was not obvious after 18h.

In addition, more strategies to improve the soluble expression of foreign proteins are mentioned in the literature [7]. Including adding special factors to the culture medium to help protein folding; Select appropriate vectors, such as vector pet32b (+) with soluble fusion label or vector with a secretory label; Some metal ions can also improve the soluble expression of protein; Change the composition of the medium or add protease inhibitors. However, due to lack of time, we did not make further exploration.

![](https://static.igem.org/mediawiki/2021/b/b7/T--ZJU-China--Results-Pic13.png)

**Figure. 2-10** Optimization of scFv expression conditions. a. The expression of total scFv protein and soluble scFv protein at different temperatures. b. The expression of total scFv protein and soluble scFv protein at different IPTG concentrations. c. The expression of total scFv protein and soluble scFv protein at different induction times. The histogram shows the relative value obtained by gray value analysis of the strip with Image J software.

**(6) Purification of soluble scFv protein from Escherichia coli**

After optimizing the expression conditions of soluble protein, we need to purify the protein. pET28a (+) vector has double His-tags, which means that we can purify it by Ni column affinity chromatography. The purification effect is shown in Fig. 2-11. Most of the miscellaneous proteins are washed off by washing a solution of low imidazole concentration, and then the adsorbed scFv protein is eluted off by eluent of high imidazole concentration. By collecting the eluent tube by tube, we get a relatively pure scFv protein. In the future, we can explore more suitable purification conditions, such as increasing the amount of washing solution or using gradient elution. The purified protein sample was added with equal volume glycerol and stored at - 20 ℃.

![](https://static.igem.org/mediawiki/2021/a/ad/T--ZJU-China--Results-Pic14.png)

**Figure. 2-11** Purification of soluble scFv protein. Lane1: DNA ladder, lane2: bacterial lysate before column passing, lane3: bacterial lysate after column passing, lane4-5: washing liquid, lane6-11: elution liquid.

**(7) Determination of concentration of scFv protein after purification**

Because the purified scFv protein is not very pure, the actual scFv protein concentration obtained by BCA protein concentration quantitative method will be very inaccurate. Therefore, we used Coomassie Blue Staining to determine the protein concentration. Use a series of standard protein samples with known concentrations and purified protein samples to run SDS-PAGE gel and stain with Coomassie brilliant blue (as shown in Fig 2-12 (a)), use Image J for gray analysis, make a standard curve (as shown in Fig. 2-12 (b)), and calculate the protein concentration represented by the target band in the sample through the standard curve. Finally, the concentration of scFv in the purified sample was 26.74ug/ml(as shown in Fig. 2-12 (c)).

![](https://static.igem.org/mediawiki/2021/4/42/T--ZJU-China--Results-Pic15.png)

**Figure. 2-12** Determination of concentration of scFv protein after purification. (a) SDS-PAGE results of standard samples and purified samples. (b) Standard curve of corresponding relationship between protein concentration and gray density. (c) Concentration calculation of scFv in purified samples.

#### Part 2.3 Identification of scFv activity

**（1）The activity of scFv protein was detected by Western blot**

After obtaining the purified soluble scFv protein, we need to verify its activity to ensure that it can recognize GPC3 antigen. Firstly, after running SDS-PAGE gel with the recombinant human GPC3 sample, we detected the recombinant GPC3 antigen with the commercially available polyclonal antibody against GPC3 and anti-GPC3 scFv respectively (as shown in Fig. 2-13 (a)). Next, we prepared HepG2 cell lysate and detected its GPC3 antigen by Western blot. The commercial polyclonal antibody against GPC3 successfully detected GPC3 core protein(full length, 66kD) and the C-terminal of the cleaved GPC3 protein(30kd) (as shown in Fig. 2-13 (b)), and scFv detected GPC3 core protein(full length, 66kD), the N-terminal of the cleaved GPC3 protein(40KD), and some glycated GPC3 proteins (as shown in Fig13 (c)). These results are consistent with those in the literature[8-10]. The full-length GPC3 can be cut into small subunits of 40KD and 30kd and can be glycated. Meanwhile, It shows that anti-GPC3 scFv has high affinity and specificity.

![](https://static.igem.org/mediawiki/2021/1/14/T--ZJU-China--Results-Pic16.png)

**Figure. 2-13** The activity of scFv protein was detected by Western blot.
(a)The detection of the recombinant GPC3 antigen with the commercially available polyclonal antibody against GPC3 and anti-GPC3 scFv respectively. (b)The detection of the endogenous GPC3 antigen of hepG2 cell with the commercially available polyclonal antibody against GPC3. (c)The detection of the endogenous GPC3 antigen of hepG2 cell with the anti-GPC3 scFv.

**（2）The detection of Kd value by ELISA**

Next, we want to make some quantitative characterization of scFv to prove that scFv has high affinity. One of the most powerful representatives is determine the Kd (ligand concentration that binds to half the receptor sites at equilibrium) and Bmax (maximum number of binding sites). Therefore, we can use hill model (Y= Bmax\*X^h / (Kd^h+X^h)) to equate the kinetics of specific binding[11].

We selected the polyclonal antibody of GPC3 and the nucleic acid aptamer specific for liver cancer cells to compare with scFv. The concentration dependence of their specific binding was characterized by ELISA. The determination results of polyclonal antibody, scFv and aptamer are shown in Fig. 2-14, Fig. 2-15 and Fig. 2-16, respectively. The Kd value of the polyclonal antibody of GPC3 is several hundreds ng/ml (as shown in Fig. 2-14), while the Kd value of scFv reaches several tens ng/ml (as shown in Fig. 2-15), which shows that the affinity of scFv for GPC3 is dozens of times that of the polyclonal antibody. Although the reported Kd values of TLS11a aptamer for hepatoma cells reached 4.51nm/ml and 7.16nm/ml for the mouse hepatoma cell line (MEAR cell line) and human hepatoma cell line LH86 [12], our results showed that TLS11a did not specifically bind to HepG hepatoma cell line (as shown in Fig. 2-16). In conclusion, the results show the high affinity of scFv.

![](https://static.igem.org/mediawiki/2021/e/eb/T--ZJU-China--Results-Pic20.png)

**Figure. 2-14** Determination of Kd value of anti-GPC3 polyclonal antibody. (a) (b) (c) Three ELISA results. (d) Parameters in hill model.

![](https://static.igem.org/mediawiki/2021/b/ba/T--ZJU-China--Results-Pic21.png)

**Figure. 2-15** Determination of Kd value of anti-GPC3 scFv. (a) (b) (c) (d) Four ELISA results. (e) Parameters in hill model.

![](https://static.igem.org/mediawiki/2021/a/ad/T--ZJU-China--Results-Pic22.png)

**Figure. 2-16** Determination of Kd value of TLS11a. (a) (b) (c) (d) Four ELISA results.

**（3）Cellular immunofluorescence**

In order to prove that anti-GPC3 scFv can bind to the membrane of HepG2 cells, we did immunofluorescence experiment. The results show that scFv has good membrane binding characteristics, which can help our recombinant adenovirus bind to the cell surface by the interaction between scFv and GPC3. In addition to the fluorescence on the membrane, the group of polyclonal antibodies also has strong fluorescence in the cytoplasm, especially in Golgi, which may be related to the nonspecific binding of polyclonal antibodies and the cleavage of GPC3 by Furin protease in Golgi [13]. Although TLS11a is considered to bind to the membrane protein of hepatoma cells, the results of cellular immunofluorescence do not show good membrane binding characteristics, which is consistent with the reported literature [14].

![](https://static.igem.org/mediawiki/2021/8/85/T--ZJU-China--Results-Pic23.png)

**Figure 2-17** Immunofluorescence of polyclonal antibody, scFv and TLS11a.

**（4） Flow cytometry**

In addition to the above experiments, we also analyzed the binding of scFv to HepG2 cells by flow cytometry. As shown in Fig. 2-18, compared with the control group, the probability of FITC positive cells in His-scFv group increased significantly and increased in a concentration dependent manner.

![](https://static.igem.org/mediawiki/2021/c/c5/T--ZJU-China--Results-Pic24.png)

**Figure 2-18** The binding of scFv to HepG2 cells was analyzed by flow cytometry.

# 3. Specific replication

#### PART 3.1 SEQUENCE PART

##### 3'UTR Sequence 226bp

< GCAGATCTAGATCTTAAAGCTTGCCACTGAAGAACTTTAGGGTTAGGGTTAGGGTTAGGGGAACAGGTAGTCTGAACACTGGGTTAGGGTTAGGGTTAGGGTTAGGGGCCAATATTTCTGTGCTGCTATTAGGGTTAGGGTTAGGGTTAGGGGAACAGGTAGTCTGAACACTGGGTTAGGGTTAGGGTTAGGGTTAGGGGAACAGGTAGTCTGAACACTGGGCTGCAGTGCA

##### Sponge Sequence 284bp

< TAAACTTGCCACTGAAGAACTCGATGCCAATTATCTGTGCTGCTATCACGAACGGTATCTGAACACTGGGAAAGTAAAGCTGCCTCTGAAGAACTGGAATGGCCAATATGCTGTGCTGCTAGTCTACGAACAGTGGCCTGAACACTGGGCTAGTGAATGGTAGTGTGAACACTGGGCACGGTAGACCTTGACTGAAGAACTAATACGCTAATATTTGTGCTGCTACTTATTAAATCTTGCCGCTGAAGAACTCGATGAAGGGTCTGAACACTGGGGCCACCATG

#### PART 3.2 Construction of 3'UTR, sponge, and null plasmid

p3UTR and pSp were generated by Genescript.

The successful construction of empty plasmid was confirmed by colonial PCR with primers 3UTR-EMPTY-F and 3UTR-EMPTY-R in Figure. 3-1 and agarose electrophoresis. The result shows in Figure. 3-2.

![](https://static.igem.org/mediawiki/2021/0/02/T--ZJU-China--Results-Pic25.png)

**Figure. 3-1** Plasmid map of 3UTR.

Primers Null\_1R and Null\_F are used to construct p3'Unull. Primers 3UTR-EMPTY-F and 3UTR-EMPTY-R are used to verify the successful construction of p3'Unull. Using p3UTR as an example here, the construction of pSp and pSp null are same with it.

![](https://static.igem.org/mediawiki/2021/thumb/c/c7/T--ZJU-China--swing.jpeg/398px-T--ZJU-China--swing.jpeg)

**Figure. 3-2** Agarose gel electrophoresis of colonial PCR product. The 226 bp sequence 3UTR was successfully removed. Lane 1: DNA ladder, lane 2: p3UTR, lane 3: p3Unull which removed 226 bp sequence 3UTR.

#### PART 3.3 miRNAs content measurement

We used Bulge-Loop primer set from Ribobio to detect miRNA expression and differential analysis based on qPCR detection technology, and we chose U6-1 as our internal reference which was a component of splicing complex. We then performed a statistical analysis of all valid experimental results. The results for ANOVA are as follows in Table. 3-1. And to further understand the miRNA content difference in different cell types, LNK test under the context of ANOVA were performed, the result of which were shown in Table. 3-2.

| Analysis of Variance Table |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | Df | Sum Sq | Mean Sq | F value | Pr(>F) | Explained% |
| Cell\_Line | 1 | 6.131 | 6.131 | 57.158 | 5.021e-08\*\*\* | 21.3628 |
| miRNA | 2 | 19.7794 | 9.8897 | 92.201 | 1.567e-12 \*\*\* | 68.91973 |
| Residuals | 26 | 2.7888 | 0.1073 |  |  | 9.717479 |

Signif. codes: 0 ‘\*\*\*' 0.001 ‘\*\*' 0.01 ‘\*' 0.05 ‘.' 0.1 ‘ ' 1

**Table. 3-1** ANOVA results of three miRNAs content in HepG2 and Hek293T. The significance was seen in miRNA content both different cell lines and miRNAs.

| LNK test |  |  |  |  |
| --- | --- | --- | --- | --- |
| Difference | pvalue | signif. | LCL | UCL |
| hek 293:195/U6 - hep G2:195/U6 | 0.917866 | 0.0002\*\*\* | 0.4833124 | 1.35241972 |
| hek 293:199/U6 - hep G2:199/U6 | 0.7182263 | 0.0046\*\* | 0.2421966 | 1.19425602 |
| hek 293:22/U6 - hep G2:22/U6 | 0.6522051 | 0.0019\*\* | 0.2635285 | 1.04088176 |

**Table. 3-2** LNK results of three miRNAs content in HepG2 and Hek293T. The significance was seen in the difference all three kinds of miRNA in both different cell lines. miRNA 195 had the largest content discrepancy in HepG2 and Hek293T.

The statistical mapping is shown as following in Figure. 3-3.

![](https://static.igem.org/mediawiki/2021/1/1d/T--ZJU-China--Results-Pic27.png)

**Figure. 3-3** mir-22,195,199 content in HepG2 and Hek293T.

This diagram shows the relative content of miRNA hsa-miR-199a, hsa-miR-195, hsa-miR-22 from HEK293T cell line and HepG2 cell line. We used reverse transcription and quantitative real-time PCR, and we choose U6-1 as our internal reference. The result shows that hsa-miR-199a, hsa-miR-195 and hsa-miR-22 express lower in HepG2 cell line than those in HEK293T cell line.

#### PART 3.4 Test of the function of the p3UTR and pSp

The data we obtained were processed the results of which are shown in Figure. 3-4.

![](https://static.igem.org/mediawiki/2021/1/1b/T--ZJU-China--Results-Pic28.png)

**Figure. 3-4** Fluorescence intensity of cells after transfection with 3u and 3ue.(3u refers to 3’UTR-pcDNA3.1-N-eGFP-C, 3u refers to 3’UTRempty-pcDNA3.1-N-eGFP-C. )

The results showed that in Hek293T transfected with p3u, the expression of eGFP was inhibited, which was not significant enough in HepG2. (p<0.1) It demonstrated that the miRNA concentration in normal cells were likely to be sufficient to inhibit the E1A.

We also designed plasmids called pRNAiU6.2-22, pRNAiU6.2-195, pRNAiU6.2-199a to increase the quantity of these miRNA in HepG2 as described previously. That was how we could know if the effect of 3UTR was actually caused by miRNA or other variables. Thus, we transfected 3u and one of these plasmids above into HepG2. The group called 3u+u6 was the controlled group, and u6 referred to pRNAiU6 without extra part. The results are shown in Figure. 3-5. Comparatively, the expressions of eGFP were all significantly decreased in HepG2 when these miRNA were replenished in cells, mir195 was the most effective one of these three. It showed that the function of 3u was related to the quantity of these miRNA, and it was reasonable to control the expression of E1A though the controlling of miRNA content.

![](https://static.igem.org/mediawiki/2021/4/40/T--ZJU-China--Results-Pic29.png)

**Figure. 3-5** Fluorescence of cells after transfection of 3u and mir199a. mir195, mir22 or u6. HepG2 transfected with 3u and certain miRNAs had significantly lower expression of eGFP compared with control group.

To further verify the specificity of the virus, we also tested the function of the designed miRNA sponges. HepG2 were transfected with 3u and sponges (sponge-pcDNA3.1-P2A-C,or sp), and the controlled group were transfected with null sponge plasmid (sponge-empty-pcDNA3.1-P2A-C, or spe), the ratio of these two plasmids was from 1:1 to 1:4. The statistical analysis results were listed below in Table. 3-3 and Table. 3-4.

|  | Df | Sum Sq | Mean Sq | F value | Pr(>F) |
| --- | --- | --- | --- | --- | --- |
| sp and spe | 1 | 1187040 | 1187040 | 20.9136 | 0.0005226\*\*\* |
| concentration gradient | 1 | 93174 | 93174 | 1.6416 | 0.2224950 |
| Residuals | 13 | 737869 | 56759 |  |  |

Signif. codes: 0 '\*\*\*' 0.001 '\*\*' 0.01 '\*' 0.05 '.' 0.1 ' ' 1

**Table. 3-3** LSD test results of the fluoresces in HepG2 and Hek293T. The significance was seen when the ratios of the two plasmids (sponge or sponge empty and 3u) were regarded as a covariate. (3u refers to 3’UTR-pcDNA3.1-N-eGFP, sponge refers to sponge-pcDNA3.1-P2A-C, and sponge empty refers to sponge-empty-pcDNA3.1-P2A-C. )

|  | Difference | pvalue | signif. | LCL | UCL |
| --- | --- | --- | --- | --- | --- |
| 3U+sp:1:1 - 3U+spe:1:1 | 384.491282 | 0.0399 | \* | 20.55030 | 748.4323 |
| 3U+sp:1:3 - 3U+spe:1:3 | 705.022404 | 0.0011 | \*\* | 341.08142 | 1068.9634 |

**Table. 3-4** LSD test results of the fluoresces in HepG2 and Hek293T. The significance was seen when the ratios of the two plasmids (3u and sponge or sponge empty ) was 1:3.(3u refers to 3’UTR-pcDNA3.1-N-eGFP, sponge or sp refers to sponge-pcDNA3.1-P2A-C, and sponge empty or spe refers to sponge-empty-pcDNA3.1-P2A-C. )

![](https://static.igem.org/mediawiki/2021/e/eb/T--ZJU-China--Results-Pic31.png)

**Figure. 3-6** Fluorescence intensity after cotransfection of 3U and Sp/Spe in Hek293T and HepG2.(3u refers to 3’UTR-pcDNA3.1-N-eGFP, sponge or sp refers to sponge-pcDNA3.1-P2A-C, and sponge empty or spe refers to sponge-empty-pcDNA3.1-P2A-C. )

According to Fig. 3-6 (a), in Hek293T, when the ratio of the 3u and the pSp or pSp null was 1:3, the expression of the eGFP was significantly lowered. Fig. 3-6 (b) shows that in HepG2, the significance was shown also when the ratio was 1:3. LSD test also showed the same results. Fig.3-7 shows the results under the microscope. Meanwhile, as shown in Table. 3-3, when the ratios of the two plasmids (sponge or sponge empty and 3u) are regarded as a covariate, the difference between the experimental group and the control group is significant, which indicates that sponges are able to increase the expressions of the eGFP. The data used in the statistical analysis is listed in the following supplementary materials. However, as the sponges also increased the expression of that genes in Hek293T, which represent normal cells, we should advocate some further improvements in the specificity of the sponge parts .

![](https://static.igem.org/mediawiki/2021/8/8b/T--ZJU-China--Results3-7.png)

**Figure 3-7** Results of the transfection.
The expression of the eGFP in HepG2 was higher than that in Hek293T. Meanwhile, when the condition was controlled, the expression of 3U increased when the quantity of pSp was three times of p3U.

We provide some [supplementary materials.](https://static.igem.org/mediawiki/2021/f/fa/T--ZJU-China--ResultsExtended.pdf)

# 4. Oncolytic Virulence

#### Part 4.1 Sequence Part

With the help of thermofisher online shRNA design tools, BLOCK-iT? RNAi Designer[15] we had designed three types of shRNA for each gene based on the information from the NCBI database. Their sequence are as following (Table .4-1) .

| **Name** | **Sequence** |
| --- | --- |
| hTERT-1-F | AATTCGCTCGTGGAGACCATCTTTCTTTCAAGAGAAGAAAGATGGTCTCCACGAGCTTTTTTG |
| hTERT-1-R | GATCCAAAAAAGCTCGTGGAGACCATCTTTCTTCTCTTGAAAGAAAGATGGTCTCCACGAGCG |
| hTERT-2-F | AATTCGGAAGAGTGTCTGGAGCAAGTTTCAAGAGAACTTGCTCCAGACACTCTTCCTTTTTTG |
| hTERT-2-R | GATCCAAAAAAGGAAGAGTGTCTGGAGCAAGTTCTCTTGAAACTTGCTCCAGACACTCTTCCG |
| hTERT-3-F | AATTCGGAAGACAGTGGTGAACTTCCTTCAAGAGAGGAAGTTCACCACTGTCTTCCTTTTTTG |
| hTERT-3-R | GATCCAAAAAAGGAAGACAGTGGTGAACTTCCTCTCTTGAAGGAAGTTCACCACTGTCTTCCG |
| VEGFA-1-F | AATTCGCAGATTATGCGGATCAAACCTTCAAGAGAGGTTTGATCCGCATAATCTGCTTTTTTG |
| VEGFA-1-R | GATCCAAAAAAGCAGATTATGCGGATCAAACCTCTCTTGAAGGTTTGATCCGCATAATCTGCG |
| VEGFA-2-F | AATTCGCGCAAGAAATCCCGGTATAATTCAAGAGATTATACCGGGATTTCTTGCGCTTTTTTG |
| VEGFA-2-R | GATCCAAAAAAGCGCAAGAAATCCCGGTATAATCTCTTGAATTATACCGGGATTTCTTGCGCG |
| VEGFA-3-F | AATTCGCGAGGCAGCTTGAGTTAAACTTCAAGAGAGTTTAACTCAAGCTGCCTCGCTTTTTTG |
| VEGFA-3-R | GATCCAAAAAAGCGAGGCAGCTTGAGTTAAACTCTCTTGAAGTTTAACTCAAGCTGCCTCGCG |

**Table. 4-1** Sequences of designed shRNA.

We found two functional \*htert\* and \*survivin\* promoter sequence in previous studies[16,17] and by searching for the \*hTERT\* and \*SURVIVIN\* genes in the NCBI database, we aligned the front part of the gene sequence to the promoter and designed corresponding primers (Table .4-2) . The primer sequences are as following.

| **Name** | **Sequence** |
| --- | --- |
| htert-F | ATTACCGCCATGCATGTTAACCAGCTGCGCTGTCGGGGCCAGG |
| htert-R | TAGCGCTAGCGGATCCATATGGCTGCCTGAAACTCGCGCCGCGA |
| survivin-F | ATTACCGCCATGCATGTTAACATTACAGGCATGCACCAC |
| survivin-R | TAGCGCTAGCGGATCCATATGAATCTGGCGGTTAATGGC |

**Table. 4-2** Primers for tumor specific promoters.

After using the human genome as a template for PCR, we obtained the corresponding promoter sequence (Table .4-4) . The CMV promoter was removed from the pSB1C3 plasmid by using linearization primers (Table .4-3) to linearize the fragments other than the promoter sequence, and then the needed promoter was reloaded into the plasmid by homologous recombination, resulting in P\*htert\*-EGFP and P\*survivin\*-EGFP plasmids. After sequencing verification, it was found that the sequence of promoter met the expectation.

| **Name** | **Sequence** |
| --- | --- |
| CMV-linear-F | GATCCGCTAGCGCTACCG |
| CMV-linear-R | ATGCATGGCGGTAATACGGT |

**Table. 4-3** Primers to delete the CMV promoter in pSB1C3 plasmid.

| **Name** | **Sequence** |
| --- | --- |
| hTERT promoter Sequence 263bp | CAGCTGCGCTGTCGGGGCCAGGCCGGGCTCCCAGTGGATTCGCGGGCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTTTCAGGCAGC |
| survivin promoter Sequence 397bp | ATTACAGGCATGCACCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAAGGTTTCACCGTGATGGCCAGGCTGGTCTTGAACTCCAGGACTCAAGTGATGCTCCTGCCTAGGCCTCTCAAAGTGTTGGGATTACAGGCGTGAGCCACTGCACCCGGCCTGCACGCGTTCTTTGAAAGCAGTCGAGGGGGCGCTAGGTGTGGGCAGGGACGAGCTGGCGCGGCGTCGCTGGGTGCACCGCGACCACGGGCAGAGCCACGCGGCGGGAGGACTACAACTCCCGGCACACCCCGCGCCGCCCCGCCTCTACTCCCAGAAGGCCGCGGGGGGTGGACCGCCTAAGAGGGCGTGCGCTCCCGACATGCCCCGCGGCGCGCCATTAACCGCCAGATT |

**Table .4-4** Sequences of functional tumor specific promoters.

**Part 4.2 shRNA part**

To have an intuitive result, we used AnnexinV to analysis the apoptosis rate of the cells. We compared the apoptosis rate between shRNA transfected groups with the scramble group( shRNA without any targeting gene) and the GADPH group( shRNA targeting GADPH, used as positive control). The result had shown higher dead rate in the functional shRNA transfected groups than in the negative control group (scramble group) . Meanwhile, they had also shown an even or higher apoptosis rate than the positive control group (GADPH group), indicating the efficiency of the shRNA. These results showed that there is at least one type of efficient shRNA for each type of gene.

![](https://static.igem.org/mediawiki/2021/a/ab/T--ZJU-China--Results-Pic32.png)

**Figure. 4-1** 48h after transfection. Using AnnexinV to see the apopotos of cells. (a): GC; (b-d):T1,T2,T3; (e):SC; (f-h):V1,V2,V3; Groups transfected with T2 and V2 had shown the highest apoptosis rate. V1-3 stands for VEGFA targeting shRNA. T1-3 stands for hTERT targeting shRNA.

![](https://static.igem.org/mediawiki/2021/b/bb/T--ZJU-China--Result-1.png)

**Figure. 4-2** Annexin V method to analyze the apoptosis of cells after the transfection of shRNA. Each of our targeting gene has a efficient shRNA for RNAi mechanism.

The qRT-PCR showed the same result from transcription level. The sramble groups showed higher mRNA expression level of \*VEGFA\* and \*hTERT\* than the processed groups, indicating the fact that our designed shRNA behaved well in the gene silence process. V1 and T1 shRNA surprisingly showed an increase of expression. This phenomenon may come from the inherent error of qRT-PCR. Actually, these two designed shRNA may had very little influence on the gene expression. The other four designed shRNA all had shown prominent reduction of gene expression, which had reached the efficient level of gene down-regulation.

![](https://static.igem.org/mediawiki/2021/c/cb/T--ZJU-China--Result-2.png)

**Figure. 4-3** For the two targeting gene that we decide to down-regulate, we had designed three types of shRNA for each. There are shRNAs which had shown a satisfying down-regulation result.

As the qRT-PCR experiment has high uncertainty, we conducted Western Blotting to analyze the down-regulation from the translational level. We had chosen the shRNA which had shown the gene expression down-regulation effect in previous experiment. Though hTERT is a type of protein expressed in cancer cells, the expression level is quite low. Therefore, this low expression had led much trouble to the detection.

We used V2,V3,T2,T3 shRNA for the experiment. The expression level of VEGFA protein had been distinctly down-regulated by V2 (V2-2) while the result of V3 is not quite clear (Figure. 4-3) . The WB result of hTERT is not quite satisfying. As the expression level of hTERT is quite low, we had to adjust to an extent in which the background signal is also high. However, it still could be recognized that T2(T2-1) shRNA had shown distinct down-regulation effect (Figure .4-4) .

These results indicated that our designed shRNA are truly efficient. In the future we may choose other types of hepatic cancer cell lines or the primary hepatocarcinoma cells for further study, hoping that a better result can be obtained.

![](https://static.igem.org/mediawiki/2021/e/ea/T--ZJU-China--Results-Pic35.png)

**Figure. 4-4** Western Blotting of VEGFA in HepG2. V2,V3 are the shRNA which previously

![](https://static.igem.org/mediawiki/2021/e/ea/T--ZJU-China--Results-Pic36.png)

**Figure. 4-5** Western Blotting of hTERT in HepG2. The expression level of the protein is low so the result may not be that satisfying.

#### part 4.3 Promoter part

The promoter expression level was shown by the intensity of the green fluorescence. The higher the intensity, the more efficient the promoter is in the situation.

As the limitation of our condition, we only got HepG2 and HEK293T cells for recognizing the expression difference. We regarded HEK293T cells as the normal cell control group, though it is not a traditional somatic cell.

We analyzed two types of promoter: P\*htert\* and P\*survivin\*. The result showed a much higher intensity of P\*survivin\* induced green fluorescence expression in HepG2 cells than in Hek293T cells, while the intensity difference of P\*htert\* induced green fluorescence is not distinctly different between these two types of cell. We made the conclusion that P\*survivin\* is an ideal promoter to trigger tumor specific expression.

P\*htert\* may also be an ideal choice because it had shown a steadily higher expression in HepG2 cells, though the excess is not large. We suppose that this is because HEK293T cells is a type of human embryonic kidney cells. \*hTERT\* gene is also expressed in human embryonic[18], so the efficiency of the promoter is also high in HEK293T cells. However, we can also say that PhTERT owns a much higher expression ability in hepatic cancer cells than in normal cells. Because compared to cells in which P\*hTERT\* functioning the most efficiently among all other normal cells, P\*hTERT\* still had shown a higher efficiency in cancer cells. Therefore, we are able to prove the tumor specificity of the P\*hTERT\*. Later we decide to use normal hepatic cell lines, L-O2, for further study in order to get a more reliable result.

All in all, we had proved the tumor specific expression of P *survivin* and P*htert*

![](https://static.igem.org/mediawiki/2021/a/aa/T--ZJU-China--Result-5.png)

**Figure. 4-6** Two types of cancer cell specific expression promoter are examined to see the differential expression in two types of cell. Survivin promoter had shown a much higher expression level in HepG2 cells, indicating that survivin promoter is an optimal choice for our design.

# 5. Virus experiments part

#### Part 5.1 Recombined and packaged the virus

We inserted our circuit into the Ad5 virus missing E1 and E3. The titer of adenovirus was detected by TCID50 method. The virus stock solution was diluted gradient by limited dilution method, from 10-1 to 10-10. Starting from the highest dilution, 100ul for each dilution was added to holes 1 to 10 of 96 well plate inoculated with hek293a cells. After 10 days, CPE phenomenon of each hole was observed under inverted microscope. The results showed that the virus titer reached 3.49 × 10^10.

#### Part 5.2 Future experiments

Since virus packaging was difficult, it was not until recently that our virus packaging was completed. We have also planned virus experiments (see our experiment) and are working hard to carry out these experiments. If there are subsequent experimental results, we will update our wiki.

# References

[1] https://www.invivogen.com/odn2216

[2] Hong, J. S., & Engler, J. A. (1996). Domains required for assembly of adenovirus type 2 fiber trimers. Journal of virology, 70(10), 7071–7078. https://doi.org/10.1128/JVI.70.10.7071-7078.1996

[3] https://zhanggroup.org/I-TASSER/

[4] https://www.rcsb.org

[5] Guo, Y., Wallace, S. S., & Bandaru, V. (2009). A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli. Protein expression and purification, 65(2), 230–237. https://doi.org/10.1016/j.pep.2008.12.013

[6] Kong, B., & Guo, G. L. (2014). Soluble expression of disulfide bond containing proteins FGF15 and FGF19 in the cytoplasm of Escherichia coli. PloS one, 9(1), e85890. https://doi.org/10.1371/journal.pone.0085890

[7] Ma, Y., He, J., &Kang, Y.(2018). Reviews on Strategies of Enhance Heterologous Protein Soluble Expression in Escherichia Coli. WORLD-SCI-TECH R&D(05),627-630. doi:10.16507/j.issn.1006-6055.2015.05.033.

[8] Nakano, K., Orita, T., Nezu, J., Yoshino, T., Ohizumi, I., Sugimoto, M., Furugaki, K., Kinoshita, Y., Ishiguro, T., Hamakubo, T., Kodama, T., Aburatani, H., Yamada-Okabe, H., & Tsuchiya, M. (2009). Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells. Biochemical and biophysical research communications, 378(2), 279–284. https://doi.org/10.1016/j.bbrc.2008.11.033

[9] Hippo, Y., Watanabe, K., Watanabe, A., Midorikawa, Y., Yamamoto, S., Ihara, S., Tokita, S., Iwanari, H., Ito, Y., Nakano, K., Nezu, J., Tsunoda, H., Yoshino, T., Ohizumi, I., Tsuchiya, M., Ohnishi, S., Makuuchi, M., Hamakubo, T., Kodama, T., & Aburatani, H. (2004). Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. Cancer research, 64(7), 2418–2423. https://doi.org/10.1158/0008-5472.can-03-2191

[10] Traister, A., Shi, W., & Filmus, J. (2008). Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. The Biochemical journal, 410(3), 503–511. https://doi.org/10.1042/BJ20070511

[11] https://www.graphpad.com/guides/prism/latest/curve-fitting/reg\_specific\_hill.htm

[12] Meng, L., Yang, L., Zhao, X., Zhang, L., Zhu, H., Liu, C., & Tan, W. (2012). Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer. PloS one, 7(4), e33434. https://doi.org/10.1371/journal.pone.0033434

[13] Haruyama, Y., & Kataoka, H. (2016). Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma. World journal of gastroenterology, 22(1), 275–283. https://doi.org/10.3748/wjg.v22.i1.275

[14]https://digital.library.txstate.edu/bitstream/handle/10877/6899/SUTTON-THESIS-2014.pdf?isAllowed=y&sequence=1

[15] http://rnaidesigner.thermofisher.com/rnaiexpress

[16]J. Yin, P. Fu, X. Wang, G. Jin, R. Xiu, Tumor-specific expression detected with the use of an expression vector driven by human telomerase reverse transcriptase gene promoter, J Lab Clin Med, 144(2004) 302-306.

[17]E. Sasso, G. Froechlich, G. Cotugno, A.M. D'Alise, C. Gentile, V. Bignone, M. De Lucia, B. Petrovic, G. Campadelli-Fiume, E. Scarselli, A. Nicosia, N. Zambrano, Replicative conditioning of Herpes simplex type 1 virus by Survivin promoter, combined to ERBB2 retargeting, improves tumour cell-restricted oncolysis, Sci Rep, 10(2020) 4307.

[18]S. Wang, C. Hu, J. Zhu, Transcriptional silencing of a novel hTERT reporter locus during in vitro differentiation of mouse embryonic stem cells, MOL. BIOL. CELL, 18(2007) 669-677.

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