iGEM 2019 — Team SYSU-Medicine

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SYSU-Medicine/Notebook

![](https://static.igem.org/mediawiki/2019/3/3c/Igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/2019/e/ee/2019_team_menu_icon.svg)

[![](https://placehold.it/350x150/ececec/cecece)](https://2019.igem.org/Team%3ASYSU-Medicine)
[HOME](https://2019.igem.org/Team%3ASYSU-Medicine)

TEAM

[Team Members](https://2019.igem.org/Team%3ASYSU-Medicine/Team)
[Collaborations](https://2019.igem.org/Team%3ASYSU-Medicine/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ASYSU-Medicine/Description)
[Design](https://2019.igem.org/Team%3ASYSU-Medicine/Design)
[Experiments](https://2019.igem.org/Team%3ASYSU-Medicine/Experiments)
[Notebook](https://2019.igem.org/Team%3ASYSU-Medicine/Notebook)
[Contribution](https://2019.igem.org/Team%3ASYSU-Medicine/Contribution)
[Results](https://2019.igem.org/Team%3ASYSU-Medicine/Results)
[Demonstrate](https://2019.igem.org/Team%3ASYSU-Medicine/Demonstrate)
[Improve](https://2019.igem.org/Team%3ASYSU-Medicine/Improve)
[Attributions](https://2019.igem.org/Team%3ASYSU-Medicine/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ASYSU-Medicine/Parts)
[Basic Parts](https://2019.igem.org/Team%3ASYSU-Medicine/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ASYSU-Medicine/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3ASYSU-Medicine/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3ASYSU-Medicine/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ASYSU-Medicine/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ASYSU-Medicine/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ASYSU-Medicine/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ASYSU-Medicine/Hardware)
[Measurement](https://2019.igem.org/Team%3ASYSU-Medicine/Measurement)
[Model](https://2019.igem.org/Team%3ASYSU-Medicine/Model)
[Plant](https://2019.igem.org/Team%3ASYSU-Medicine/Plant)
[Software](https://2019.igem.org/Team%3ASYSU-Medicine/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=SYSU-Medicine)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SYSU-Medicine/Experiments

<!doctype html>

SYSU-Medicine

* [Home](https://2019.igem.org/Team%3ASYSU-Medicine)
* Project
  + [Description](https://2019.igem.org/Team%3ASYSU-Medicine/Description)
  + [Design](https://2019.igem.org/Team%3ASYSU-Medicine/Design)
  + [Results](https://2019.igem.org/Team%3ASYSU-Medicine/Results)
  + [Experiments](https://2019.igem.org/Team%3ASYSU-Medicine/Experiments)
  + [Inspiration](https://2019.igem.org/Team%3ASYSU-Medicine/Inspiration)
* Model
  + [Overview](https://2019.igem.org/Team%3ASYSU-Medicine/Model)
  + [M1 Cultivation](https://2019.igem.org/Team%3ASYSU-Medicine/M1_Cultivation)
  + [Injection
    System](https://2019.igem.org/Team%3ASYSU-Medicine/Injection_System)
  + [T. E. S.](https://2019.igem.org/Team%3ASYSU-Medicine/TES)
  + [Future](https://2019.igem.org/Team%3ASYSU-Medicine/Future)
* Human Practices
  + [Overview](https://2019.igem.org/Team%3ASYSU-Medicine/Human_Practices)
  + [Integrated
    Practices](https://2019.igem.org/Team%3ASYSU-Medicine/Integrated_Practices)
  + [Education &
    Engagement](https://2019.igem.org/Team%3ASYSU-Medicine/Public_Engagement)

* Parts
  + [Parts Overview](https://2019.igem.org/Team%3ASYSU-Medicine/Parts)
  + [Basic
    Parts](https://2019.igem.org/Team%3ASYSU-Medicine/Parts#Basic_Part)
  + [Composite
    Parts](https://2019.igem.org/Team%3ASYSU-Medicine/Parts#Composite_Part)
  + [Part
    Collection](https://2019.igem.org/Team%3ASYSU-Medicine/Parts#Part_Collection)
* [Safety](https://2019.igem.org/Team%3ASYSU-Medicine/Safety)
* [Awards](https://2019.igem.org/Team%3ASYSU-Medicine/Medal)
  + [Judging](https://igem.org/2019_Judging_Form?team=SYSU-Medicine)
* Team
  + [Team Members](https://2019.igem.org/Team%3ASYSU-Medicine/Team)
  + [Collaborations](https://2019.igem.org/Team%3ASYSU-Medicine/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ASYSU-Medicine/Attributions)

# EXPERIMENT

# Protocol

* Molecular

  ### [Protocol for Agarose Gel Electrophoresis](#step-1)

  1.Weigh 0.8g (0.4g) agarose powder with Electronic scale, and get
  80ml (40ml) 1X TAE buffer and put them together into a flask and
  mix them to get 1% solution;

  2.Melt the mixture in a microwave with a middle-high heat for
  about 2 min until the solution becomes clear;

  3.Put 8 (4) ul EB solution into the mixture prepared above;

  4.Put the flask under the flowing water to cool it to about
  40-50°C(it’s okay when you don’t feel hard to hold it）, and pour
  the whole solution into the gel casting tray with appreciate
  comb;

  5.Let the gel cool until it is solid(it takes at least 30 min)

  6.Pull out the comb carefully and avoid damaging the Gel;

  7.Place the gel in the electrophoresis chamber;

  8.Pipette DNA samples mixed with appreciate amount of loading
  buffer and dye (GeneFinder) into wells on the gel;

  9.Run the gel at 120V for about half an hour;
* ### [Protocol for cDNA RT reactions](#step-2)

  1.Pipette 10μL of RNA sample and 1.5μL oligo(dT)15 into each
  individual tube.

  2.Put the heating block ready and set the temp to 70℃.And put the
  tube into the heating block for 5min.

  3.Put the heated tube on the icebox and cool down quickly for
  another 5min.

  4.Centrifuge for a short time to spin down the contents and to
  eliminate any air bubbles.

  5.Pipette 4μL 5xBuffer、2μL Mgcl2、1μL dNTP Mix (10 mM)、0.5μL
  Ribonuclease Inhibitor、1μL RTase together into the tube.

  6.Vortex for a short time to completely mix the sample.

  7.Reset the heating blocker to 25℃ and put the tube in the
  blocker for 5 min.

  8.Change the temp to 42℃ for an hour.
* ### [Protocol for colony PCR](#step-3)

  1.Put Forward primer 2.5μl and Reverse primer 2.5μl into 18μl
  ddH2O2.

  2.Put the system above into 25μl Q5 High-Fidelity DNA Polymerase.

  3.Mix them all together.

  4.After preparation of the reaction mix, pipette 2μl Bacterial
  liquid and dip in the mix. The reaction condition settings are
  as follows:

  | Temperature | Time | Parameter |
  | --- | --- | --- |
  | 98℃ | 30s |  |
  | 98℃ | 10s | 35 |
  | 66℃ | 30s | 35 |
  | 72℃ | 2min | 35 |
  | 72℃ | 2min |  |
* ### [Protocol for preparation of LB Agar plates](#step-4)

  #### Materials

  LB Agar、Distilled water、Erlenmeyer

  #### Protocol

  1.Add LB agar 15g to 1L.

  2.Add 1L distilled water into Erlenmeyer

  3.Cover Erlenmeyer with aluminum foil

  4.Autoclave liquid program

  5.Wait until the LB cools down and add Ampicillin (1:1000 → if
  500ml then add 500ul)

  6.Pour the liquid into petri dishes (fill only ¾ of the plate and
  shake it until the liquid covers the surface)

  7.Wait 15-20 min and put the plates upside down in 4℃
* ### [Protocol for preparation of liquid LB](#step-5)

  #### Materials

  LB broth and Distilled water

  #### Protocol

  1.Add 25g of LB broth to 1 Liter.

  2.Add 1 Liter/ 100ml/ 200ml of distilled water.

  3.Cover Erlenmeyer with aluminum foil.

  4.Autoclave-liquid program.

  5.Add antibiotic only after cold down.
* ### [Protocol for total RNA Isolation](#step-6)

  1.Prepare M1 virus samples for isolation of RNA.

  2.pipette 100μL samples to a 1.5ml centrifuge tube.

  3.Add 300μL of lysis solution (added with 800μL 1-thioglycerol
  already) to each sample. Mix the samples by vigorous shaking or
  vortexing for 30 seconds, and then centrifuge for 30s.

  4.Stand for 5min at room temperature.

  5.Add 300μL of RNA diluent to each sample. Mix the samples by
  vigorous shaking or vortexing for 30 seconds, and then
  centrifuge for 30s.

  6.Stand for 5min at room temperature.

  7.Add 350μL of Anhydrous ethanol to each sample. Mix the samples
  by vigorous shaking or vortexing for 30 seconds, and then
  centrifuge for 30s.

  8.Stand for 5min at room temperature.

  9.Transfer the mixture (700μL at most per time) to the
  Centrifugal column (provided with kit), centrifuge at 12000rpm
  for 1min and discard filtrate.

  10.Add 600μL RNA lotion (added with 70ml Anhydrous ethanol
  already), centrifuge at 12000rpm for 1min and discard filtrate.

  11.Add 50μL DNA enzyme I incubation solution (5μL DNA enzymeⅠand
  DNA enzymeⅠbuffer added into 40μL nuclease-free water) to the
  center of the adsorption film, sand for 15 min at room temp.

  12.Add 600μL RNA lotion (added with 70ml Anhydrous ethanol
  already), centrifuge at 12000rpm for 1min and discard filtrate.

  13.Repeat step12

  14.Empty the collection tube and recentrifuged the column for
  2min at 12000rpm.

  15.Transfer the centrifuge column to the Elution tube, add 50μL
  nuclease-free water to the center of the adsorption film and
  stand for 2min at room temperature.

  16.Centrifuge the tube at 12000 rpm for 1min and stored the
  eluted RNA at -80℃.
* ### [Protocol for transformation](#step-7)

  1.Take out the competent cell dh5α from -80℃ and thaw it on the
  ice for 15min.

  2.Add the target plasmid into competent cell (total amount
  greater than 1ng) and put it on the ice for 30min.

  3.Set the heating block to 42℃ and put the sample on the heating
  block for 30-90s.

  4.Put the sample on the ice for 5min and then coated plate.
* ### [Protocol for Gel and PCR Clean-Up](#step-8)

  #### Materials

  •1.5ml microcentrifuge tubes

  •ethanol (95%)

  •agarose gel (standard or low-melt; only for gel puriﬁcation)

  •1X TAE or TBE electrophoresis buﬀer (only for gel puriﬁcation)

  •50–65°C heating block (only for gel puriﬁcation)

  #### Protocol

  1.Place one SV Minicolumn in a Collection Tube for each dissolved
  gel slice or PCR ampliﬁcation.

  2.Transfer the dissolved gel mixture or prepared PCR product to
  the SV Minicolumn assembly and incubate for 1 minute at room
  temperature.

  3.Centrifuge the SV Minicolumn assembly in a microcentrifuge at
  16,000 × g (14,000rpm) for 1 minute. Remove the SV Minicolumn
  from the Spin Column assembly, and discard the liquid in the
  Collection Tube. Return the SV Minicolumn to the Collection
  Tube.

  4.Wash the column by adding 700µl of Membrane Wash Solution,
  previously diluted with 95% ethanol (Section 4.A), to the SV
  Minicolumn. Centrifuge the SV Minicolumn assembly for 1 minute
  at 16,000 × g (14,000rpm). Empty the Collection Tube as before,
  and place the SV Minicolumn back in the Collection Tube. Repeat
  the wash with 500µl of Membrane Wash Solution, and centrifuge
  the SV Minicolumn assembly for 5 minutes at 16,000 × g.

  5.Remove the SV Minicolumn assembly from the centrifuge, being
  careful not to wet the bottom of the column with the ﬂowthrough.
  Empty the Collection Tube, and recentrifuge the column assembly
  for 1 minute with the microcentrifuge lid open (or oﬀ) to allow
  evaporation of any residual ethanol.

  6.Carefully transfer the SV Minicolumn to a clean 1.5ml
  microcentrifuge tube. Apply 50µl of Nuclease-Free Water directly
  to the center of the column without touching the membrane with
  the pipette tip. Incubate at room temperature for 1 minute.
  Centrifuge for 1 minute at 16,000 × g (14,000rpm).

  7.Discard the SV Minicolumn, and store the microcentrifuge tube
  containing the eluted DNA at 4°C or –20°C.
* ### [Protocol for plasmid linearization](#step-9)

  1.Get the system ready as follows:

  | Reagent | Amount(μL) |
  | --- | --- |
  | 10× FastDigest Buffer | 10 |
  | XbaI | 10 |
  | pBR-M1-c6v2-XXXXX | 80 |

  2.Divide the system into two tubes equally.

  3.Put those tubes into PCR instrument and set the temp to 37℃ to
  incubate for 1h.

  4.Add 2.5μL 1xProteinase k and 5μL 10% SDS to each tube and put
  them at 50 ℃ to react for 30min.
* ### [Protocol for transcription in vitro](#step-10)

  1.Prepare the transcription system as follows:

  | Reagent | Amount（μl） |
  | --- | --- |
  | SP6 Transcription 5X Buffer | 8 |
  | rNTPs（100 mM）（A+C+U+G） | 2+2+2+1.2 |
  | Linearized DNA template (1~2 μg）+ ddH2O | 16.8 |
  | Ribo m7G Cap Analog，40 mM | 4 |
  | Enzyme Mix | 4 |
  | Total Volume | 40 |

  2.React at 37℃ for 1h.

  3.Add 2μL RQ1 RNase-Free DNase（1U/ul）and react at 37℃ for 15min.
* Cell

  ### [Protocol for Virus Transduction to Vero cells](#step-11)

  1.Prepare Vero cells one day in advance (T25，400,000 cells/well).

  2.Add 125μL Opti-MEM Culture medium and 3.75μL MessengerMAX in a
  centrifuge tube. Then prepare another centrifuge tube added with
  125μL and RNA sample.

  3.Fully mix these two tubes solution and drip onto Vero cells.
* ### [Protocol for MTT assay](#step-12)

  1.Prepare a 12 mM MTT stock solution by adding 1 mL of sterile
  PBS to one 5 mg vial of MTT (Component A). Mix by vortexing or
  sonication until dissolved.

  2.Add 10 mL of 0.01 M HCl to one tube containing 1 gm of SDS
  (Component B). Mix the solution gently by inversion or
  sonication until the SDS dissolves.

  3.For adherent cells, remove the medium and replace it with 100
  µL of fresh culture medium. For non-adherent cells, centrifuge
  the microplate, pellet the cells, carefully remove as much
  medium as possible and replace it with 100 µL of fresh medium.

  4.Add 10 µL of the 12 mM MTT stock solution (prepared in step 1)
  to each well. Include a negative control of 10 µL of the MTT
  stock solution added to 100 µL of medium alone.

  5.Incubate at 37°C for 4 hours. At high cell densities
  (>100,000 cells per well) the incubation time can be
  shortened to 2 hours.

  6.Add 100 µL of the SDS-HCl solution (prepared in step 2) to each
  well and mix thoroughly using the pipette.

  7.Incubate the microplate at 37°C for 4 hours in a humidified
  chamber. Longer incubations will decrease the sensitivity of the
  assay.

  8.Mix each sample again using a pipette and read absorbance at
  570 nm.

  9.After labeling the cells with MTT, as described above, remove
  all but 25 µL of medium from the wells. For non-adherent cells
  it may be necessary to first centrifuge the plates to sediment
  the cells.

  10.Add 50 µL of DMSO to each well and mix thoroughly with the
  pipette.

  11.Incubate at 37°C for 10 minutes.

  12.Mix each sample again and read absorbance at 540 nm not 570
  nm, as above.
* Animal

  ### [Protocol for Animal experiment](#step-13)

  1.Prepare c57 mice and balb/c nude mice by implanting tumor
  subcutaneously and injecting engineered virus through tail vein.

  2.Observing the distribution of the virus in vivo through in vivo
  imaging system.

  3.Observing body weight and tumor size of mice after continuous
  administration.

![](https://static.igem.org/mediawiki/2019/1/13/T--SYSU-Medicine--footer_logo.png)

![](https://static.igem.org/mediawiki/2019/f/fe/T--SYSU-Medicine--five_logo.png)

Address: No.74 Zhongshan Rd.2, Guangzhou, P.R. China

ZipCode: 510080

E-mail: sysumedicine2019@163.com

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SYSU-Medicine/Results

<!DOCTYPE html>

SYSU-Medicine

* [Home](https://2019.igem.org/Team%3ASYSU-Medicine)
* Project
  + [Description](https://2019.igem.org/Team%3ASYSU-Medicine/Description)
  + [Design](https://2019.igem.org/Team%3ASYSU-Medicine/Design)
  + [Results](https://2019.igem.org/Team%3ASYSU-Medicine/Results)
  + [Experiments](https://2019.igem.org/Team%3ASYSU-Medicine/Experiments)
  + [Inspiration](https://2019.igem.org/Team%3ASYSU-Medicine/Inspiration)
* Model
  + [Overview](https://2019.igem.org/Team%3ASYSU-Medicine/Model)
  + [M1 Cultivation](https://2019.igem.org/Team%3ASYSU-Medicine/M1_Cultivation)
  + [Injection
    System](https://2019.igem.org/Team%3ASYSU-Medicine/Injection_System)
  + [T. E. S.](https://2019.igem.org/Team%3ASYSU-Medicine/TES)
  + [Future](https://2019.igem.org/Team%3ASYSU-Medicine/Future)
* Human Practices
  + [Overview](https://2019.igem.org/Team%3ASYSU-Medicine/Human_Practices)
  + [Integrated
    Practices](https://2019.igem.org/Team%3ASYSU-Medicine/Integrated_Practices)
  + [Education &
    Engagement](https://2019.igem.org/Team%3ASYSU-Medicine/Public_Engagement)

* Parts
  + [Parts Overview](https://2019.igem.org/Team%3ASYSU-Medicine/Parts)
  + [Basic Parts](https://2019.igem.org/Team%3ASYSU-Medicine/Parts#Basic_Part)
  + [Composite
    Parts](https://2019.igem.org/Team%3ASYSU-Medicine/Parts#Composite_Part)
  + [Part
    Collection](https://2019.igem.org/Team%3ASYSU-Medicine/Parts#Part_Collection)
* [Safety](https://2019.igem.org/Team%3ASYSU-Medicine/Safety)
* [Awards](https://2019.igem.org/Team%3ASYSU-Medicine/Medal)
  + [Judging](https://igem.org/2019_Judging_Form?team=SYSU-Medicine)
* Team
  + [Team Members](https://2019.igem.org/Team%3ASYSU-Medicine/Team)
  + [Collaborations](https://2019.igem.org/Team%3ASYSU-Medicine/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ASYSU-Medicine/Attributions)

# RESULTS

* ![](https://static.igem.org/mediawiki/2019/1/14/T--SYSU-Medicine--Results_%281%29.svg)

  #### [Determination of the infection and oncolytic ability of M1 virus to different tumor cell lines](#collapse-1)

  ![](https://static.igem.org/mediawiki/2019/1/1c/T--SYSU-Medicine--Result-fig1.jpg)

  Figure 1
  (A) Cell viability after infection of M1 virus.
  (B) Cell expressed eGFP after infection of M1 virus.Images were obtained 24 hours after infection.
  (C) Infection rate were determined 24 hours after infection of M1 virus.
  Though M1 virus has been shown to be infectious to different types of tumors include human and mouse cell lines,it has only limited killing effects in resistant cell lines such as A549,Hct8 and Bxpc3 in vitro.Wild type M1 virus has been engineered to express green fluorescent protein in infected cells(M1-GFP),and the percentage of green fluorescent cells can be used as a marker of infection rate and could be detected by flow cytometry.Cell viability assay was determined using an MTT kit.The infection rate of resistant cell lines could meet the requirements of enzyme expressing cells of nitroreductase mediated podrug-enzyme system.
* ![](https://static.igem.org/mediawiki/2019/9/96/T--SYSU-Medicine--Results_%282%29.svg)

  #### [Determination of the targeting ability of M1 virus to transplanted tumor](#collapse-2)

  ![](https://static.igem.org/mediawiki/2019/d/d3/T--SYSU-Medicine--Result-fig2.jpg)

  Figure 2 M1-iRFP showed targeting ability to tranplanted tumor on nude mice.(Dpi:Days post infection)

  Wild type M1 virus has been engineered to express iRFP in infected cells(M1-iRFP),which can be used for bioluminescence,in infected cells.In vivo without a complete immune system,intravenous injection of M1 virus showed both high tumor targeting ability in mouse model transplanted with Hep3B cells.
* ![](https://static.igem.org/mediawiki/2019/3/34/T--SYSU-Medicine--Results_%283%29.svg)

  #### [Establishment of M1 virus strain expressing nitroreductase](#collapse-3)

  ![](https://static.igem.org/mediawiki/2019/d/d4/T--SYSU-Medicine--Result-fig3.jpg)

  Figure 3
  (A) Plasmid for M1-GFP had been engineered to replaced the eGFP sequence with human codon optimized nitroreductase sequence from E.coli K12.
  (B) Expression of nitroreductase were determined in vero cells, b16 and A549 cell line.
  The nitroreductase coding sequence from E.coli was introduced into the genome of M1 virus to replace the green fluorescent protein sequence(M1-NR),this process was achieved by enzyme cutting and T4 ligation of target fragment at the restriction ezyme cutting sites of MfeI and XhoI.We used anti-flag antibody to verify the expression of nitroreductase by western blot 24 hours post M1-NR infection (MOI=1).And the expression of eGFP was determined 24 hours post M1-GFP infection as control(MOI=1).
* ![](https://static.igem.org/mediawiki/2019/c/c2/T--SYSU-Medicine--Results_%284%29.svg)

  #### [M1 virus mediated prodrug-enzyme system showed better antitumor activity](#collapse-4)

  ![](https://static.igem.org/mediawiki/2019/3/33/T--SYSU-Medicine--Result-fig4.jpg)

  Figure 4 Cell viability assay at different time points to verify the lethality of M1-NR together with CB1954.
  MTT assay showed a better anti-tumor ability when M1-NR and CB1954 were added into the culture medium at the same time than adding M1-NR or CB1954 independently to the culture system.Considering anti-tumor ability of this prodrug-enzyme system is closely related to the expression level of nitroreductase,we infected cells at moi=10.
* ![](https://static.igem.org/mediawiki/2019/f/f2/T--SYSU-Medicine--Results_%285%29.svg)

  #### [Prodrug derived from actinomycin D(ActD) is a potential optimizing tool for our system](#collapse-5)

  ![](https://static.igem.org/mediawiki/2019/2/27/T--SYSU-Medicine--Result-fig6a.jpg)

  Figure 5 (A) Screening the drugs that may promote M1 infection.

  ![](https://static.igem.org/mediawiki/2019/b/b0/T--SYSU-Medicine--Result-fig6b.jpg)

  Figure 5 (B) Cell treated with actinomycin D showed higher infection rate.

  ![](https://static.igem.org/mediawiki/2019/4/4f/T--SYSU-Medicine--Result-fig6c.jpg)

  Figure 5 (C) Synergistic killing of actinomycin D and virus on tumor cell lines.

  ![](https://static.igem.org/mediawiki/2019/6/6f/T--SYSU-Medicine--Result-fig6d.jpg)

  Figure 5 (D) Prodrug derived from actinomycin D had been shown to convert to its effective form in the presence of nitroreductase.

  We selected several drugs with similar structure and transcriptional blocking function to determine their power to promote M1-GFP infection.The infection rate was determined by flow cytometry 24 hours post infection on A549(MOI=1).All the drugs were added into the culture medium 9 hours post infection at a concentration of 1uM.For further detection of infection rate improved by actinomycin D ,images were captured 24 hours after M1-GFP infection and cell were treated with 0.3ug/ml actinomycin D 9 hours after infection.Infection rates were determined by flow cytometry 36 hours post infection.We hypothesized that the presence of prodrug of actinomycin D could be a stressful environment which could help us to screening out viruses with stronger prodrug converting ability.

In addition We have inserted the riboswitches into M1 virus genome, but we failed to test its function due to limited time. However, we did have detected the ribozyme activity in [parts](https://2019.igem.org/Team%3ASYSU-Medicine/Parts)

### Conclusion

We first evaluated the infection and oncolytic ability of M1 virus to different tumor cell lines in vitro, as well as its targeting ability to transplanted tumor in vivo. We then constructed M1 virus strain expressing nitroreductase to verify the significant effect of prodrug enzyme system CB1954/NTR and M1 combination on the anti-tumor activity. Furthermore, we verified that the chemotherapy drug actinomycin D can in turn promote M1 infection on a variety of tumor cell lines. Therefore, if we can combine the advantages of the two, this would be an ideal M1 prodrug enzyme system. In addition, we have also inserted riboswitches to edit M1 to be more controllable as a tool of synthetic biology.

![](https://static.igem.org/mediawiki/2019/1/13/T--SYSU-Medicine--footer_logo.png)

![](https://static.igem.org/mediawiki/2019/f/fe/T--SYSU-Medicine--five_logo.png)

Address: No.74 Zhongshan Rd.2, Guangzhou, P.R. China

ZipCode: 510080

E-mail: sysumedicine2019@163.com