iGEM 2019 — Team SYSU-CHINA

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# Team:SYSU-CHINA/Notebook

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

# Notebook

* [Record](#t)

# Molecular Experiment Record

---

### 2019.5.18

Streak E.coli containing pL7Ae and pL7AeMUT onto the LB plate. After 14 hours, pick a single colony and patch it to a new plate.

### 2019.5.19

1. Colony PCR

Primers: L7Ae-F-KpnI and L7Ae-R-BamHI primers

Enzyme: Taq

2. Detection by agarose gel electrophoresis: negative.

Analysis: Primer design error, the restriction enzyme cutting site is in the 3' end of the primer

### 2019.5.26

1. Cultivate the bacteria to be ready for extracting pL7Ae and pL7AeMUT:

Escherichia coli containing pL7Ae and pL7AeMUT was inoculated in 3L Amp+ LB medium at 37 °C overnight.

2. Screen positive bacteria:

Streak E.coli containing pL7Ae and pL7AeMUT on the LB plate. After 14 hours, pick a single colony and patch it to a new plate. After 12 hours of incubation, the patch plate was stored at 4 ° C for detection.

### 2019.5.27

1. Extraction of plasmid: Plasmid pL7Ae and pL7AeMUT were extracted using Omega E.Z.N.A Plasmid Mini Kit.

2. DNA concentration determination: L7Ae (69.1 ng / μL) ; L7AeMUT (54.3 ng / μL)

### 2019.5.31

1. PCR amplification:

L7Ae and L7AeMUT were amplified by PCR using Phata enzyme and BamHI/XhoI enzyme cutting site and Myc tag were introduced. The primers are as follows:

L7Ae-Fw-BamHI: 5'-AAGGATCCATCATATGCGGCCGCTTATGTACGTGAGATTTGAGG-3’

L7Ae-Re-Myc-XhoI:5'-CTCGAGTTAATTCAGATCCTCTTCTGAGATGAGTTTTTGTTCGAAGGGCCCTCTAGACTCCTTCTGAAGGCCTTTAATCTT-3’

2. Identification by agarose gel electrophoresis: the amount of product should be 440 bp, but the band is too wide, so consider increasing the annealing temperature and re-PCR

### 2019.6.1

1. PCR amplification under improved conditions: Amplify L7Ae and L7AeMUT using the above primers, and increase the annealing temperature to 58 °C for higher amplification specificity.

2. Agarose gel electrophoresis: higher band specificity was shown.

3. Gel extraction:
The target product was extracted with omega Gel Extraction Kit to obtain L7Ae-Myc and L7AeMUT-Myc DNA fragments. Store at 4 ° C.

### 2019.6.2

1.DNA concentration detection: UV spectrophotometer detects the concentration of extracted L7Ae-Myc and L7AeMUT-Myc fragments:
L7Ae-Myc: 169 ng/μL; L7AeMUT-Myc: 116 ng/μL

2. Digestion: double enzyme digestion of pcDNA3.1, L7Ae-Myc, L7AeMUT-Myc. The systems are as follows (unit μL):

![](https://static.igem.org/mediawiki/2019/d/d0/T--SYSU-CHINA--E1.jpg)![](https://static.igem.org/mediawiki/2019/2/2f/T--SYSU-CHINA--E2%282%29.jpg)

React for two hours at room temperature. Heat in a 70 °C water bath for 10 min to inactivate enzyme.

2. Separation of the digested product by agarose gel electrophoresis:

Only the pcDNA3.1 product was seen, and no L7Ae-Myc and L7AeMUT-Myc fragments were found, which were suspected to be lost during the gel recovery process.

3. Re-PCR amplification

The L7Ae and L7AeMUT were re-amplified using primers L7Ae-Fw-BamHI and L7Ae-Re-Myc-XhoI, and the annealing temperature was set to 57 ° C to increase the amplification specificity. The system is as follows (unit μL):

![](https://static.igem.org/mediawiki/2019/9/93/T--SYSU-CHINA--E3.jpg)

4. Detection of recovered product by agarose gel electrophoresis

Products: L7Ae-Myc and L7AeMUT-Myc both showed the target band

5. Gel extraction:

The target product was extracted with omega Gel Extraction Kit to obtain L7Ae-Myc (GSF) and L7AeMUT-Myc (GSF) DNA fragments, which were stored at 4 ° C.

6. DNA concentration detection: UV spectrophotometer detects the extracted L7Ae-Myc (GSF) and L7AeMUT-Myc (GSF) fragments:

L7Ae-Myc (GSF): 160 ng/μL; L7AeMUT-Myc (GSF): 110 ng/μL

7. Colony PCR

Use the 5.26 preserved patch plate, pick L7Ae and L7AeMUT positive for colony PCR amplification, using primers:

L7Ae-Text-Fw: ATGTACGTGAGATTTGAGGTTC

L7Ae-Text-Re: TTACTTCTGAAGGCCTTTAATCTT

8. Agarose gel electrophoresis test:

All positive

9. Amplify positive clones to be ready for preservation

L7Ae and L7AeMUT positive clones were inoculated to 5 mL of Amp+ (1000x) LB liquid medium, and shaken at 37 ° C overnight.

### 2019.6.3

1. Conservation of DH5α-L7Ae and DH5α-L7AeMUT

2 tubes of DH5α-L7Ae and DH5α-L7AeMUT were stored in 15% glycerol and placed at -80 °C

2. The overnight digestion product was collected and inactivated in a water bath at 70 ° C for 10 min. Only the pcDNA3.0 band could be seen. No gene fragment band.

3. Improve digestion condition

According to NEB protocol, the enzyme should be digested for 5-15 minutes. Considering the lower enzyme digestion efficiency of XhoI, the enzyme digestion was carried out in the same system for 25 minutes at room temperature and 5 minutes at 65 °C.

4. Column extract product

Directly extract the digested products L7Ae-Myc-BamHI-XhoI and L7AeMUT-Myc-BamHI-XhoI (undetermined concentration) using Omega Gel Extraction Kit

5. T4 ligase connection

The T4 ligase was used to link the gene to the vector. Since the concentration was not determined, the following system did not conform to the gene: vector >3:1 recommendation.

The system is as follows (unit μL):

![](https://static.igem.org/mediawiki/2019/3/38/T--SYSU-CHINA--E4.jpg)

After 1 h incubation at room temperature, the reaction was terminated by inactivation at 65 ° C for 5 min. Product stored at 4 ° C

### 2019.6.4

1. Column extract product

Extract the enzymatically linked products pcDNA3.1-L7Ae-Myc-BamHI-XhoI and pcDNA3.1-L7AeMUT-Myc using Omega Gel Extraction Kit

2. Escherichia coli DH5α plasmid transformation

Two tubes of DH5α competent were taken and the ligated plasmids pcDNA3.1-L7Ae-Myc-BamHI-XhoI and pcDNA3.1-L7AeMUT-Myc were transformed, respectively.
The system is as follows:

![](https://static.igem.org/mediawiki/2019/c/c5/T--SYSU-CHINA--EXP4.jpg)

Mix and incubate for 30 min on ice. Heat shock at 42 °C for 70 s.

After adding 1L LB medium, resuscitate at 37 ° C for 1 h

Centrifuge at 10000xg for 2min, discard the supernatant, add 200ml and resuspend
The suspension was coated with Amp+ resistant LB plate and cultured at 37 °C.

### 2019.6.5

No positive colonies after E. coli transformation

### 2019.6.8

1. Improved enzyme digestion conditions

Re-cut and adjust the digestion time to 15 min.
Reset the enzyme system and increase the amount of carrier added to 5μl.
37 ° C enzyme ligation for 1 h.

2. Improved transformation conditions

The enzyme-linked product was directly added to the DH5α competent state without column purification, and the enzyme-linked product was added to 10 μl.
Ice incubation time extended to 1h.
Add ampicillin to LB liquid medium during resuscitation.

### 2019.6.9

1. 4 plates produce positive

Analysis: It can be seen that the previous unsuccessful conversion problem is not in the plasmid, but in the conversion process and conditional issues.

Patch positives expanded for 5h.

2. Identification of positives by primer L7Ae-Text-Fw and L7Ae-Text-Re colony PCR

The test was fully positive and the plasmid was successfully constructed.

Inoculate and overnight shaking.

### 2019.6.10

1. Endotoxin-free extract plasmid endo-pcDNA3.1-L7AeMUT and endo-pcDNA3.1-L7Ae

2. Mr. He Lei provided E.coli containing the pcDNA-EGFP plasmid. Inoculate it in LB to prepare for extracting plasmids.

### 2019.6.14

1. DNA concentration detection

UV spectrophotometer was used to detect endo-pcDNA3.1-L7AeMUT and endo-pcDNA3.1-L7Ae concentrations:

endo-pcDNA3.1-L7AeMUT: 264.35 ng/μL; endo-pcDNA3.1-L7Ae: 209.35 ng/μL

### 2019.6.24

1. Plasmid extraction

pTK295, pEF1α\_reTA3, pTRE\_EGFP2, pAdeasy: extract plasmid (endotoxin-free)

pShuttle, pAdTrack, pAVV-Ubc, LSBr5and3: extract plasmid (not endotoxin-free)

### 2019.6.29

1. Gel extraction of 1xKt-EGFP and 2xKt-EGFP

2. UV spectrophotometer to measure concentration

1xKt-EGFP: 43.6 ng/uL ; 2xKt-EGFP: 64.3ng/uL

3. Inoculate DH5α-pcDNA3.1 in LB medium. Shake overnight.

### To be continued……

#### To know more about our experiment arrangement and work flow, please refer to these intact experiment records below.

[Adenovirus Experiment Record](https://static.igem.org/mediawiki/2019/4/47/T--SYSU-CHINA--AER.pdf)

[Cell Experiment Record](https://static.igem.org/mediawiki/2019/1/15/T--SYSU-CHINA--CER.pdf)

[Molecular Experiment Record](https://static.igem.org/mediawiki/2019/9/9a/T--SYSU-CHINA--MER.pdf)

---

\


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# Team:SYSU-CHINA/Experiments

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# Experiments

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

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:SYSU-CHINA/Results

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# Results

* [Results](#t)
* [Tet-on swtich](#c2)
* [Carrier and killer：Virus system](#c3)
* [Composite system work](#cx)
* [Conclusion](#c5)

# Results

---

We wonder if our system works as expected and has the potential to be applied to real-world scenarios. So we demonstrate the feasibility of this project through verification of each independent system, serial effect verification and modeling analysis.

# What had we achieved?

1. Sensor: Identified tumor miRNA profile through miRNA sensor

2. Switch: Verified the response sensitivity of the system switch

3. Carrier: Reorganized and packaged the virus

4. Killer: Characterized the efficacy of genes that turn on the virus to kill tumors

5. The system works normally after being connected in series

6. Predicated and improved the working condition of oncolytic virus in tumor treatment by modeling

### Differentiation of miRNA profiles using miRNA logic gates

#### L7Ae K-turn system

We engineered the L7Ae protein as an intermediate inhibitor in the miRNA sensor design. (Figure. 2a) By testing we found that it does not have obvious cytotoxicity (Figure.2 c,d)

![]( https://static.igem.org/mediawiki/2019/6/60/T--SYSU-CHINA--Demonstrate-L7Ae.png
)

**Figure 2**This picture shows the construction and characterization of L7AeO. **a.** According to the 3D structural analysis of the protein, the Thr on the α-helix interacting with the substrate is mutated to Pro by overlap PCR. **b.** 3D structure of L7Ae combined with ligand from PDB **c.**L7Ae/L7AeO cytotoxicity results microscopy **d.**Semi-quantitative determination of cytotoxicity by detecting cell reducing ability by MTS staining.

Then we quantitatively and qualitatively characterize the working ability of this system. The result shows that the mutant was weakened as expected but still functions in high concentration. Meanwhile, the 1000ng/ml L7Ae plasmid can depress the translation of mRNA with an efficiency of 84%. (Figure.3a) At the same time, we determined the relationship between the number of K-turn structures on mRNA and the inhibition efficiency. Only two Kt can achieve significant inhibition effect, and the inhibition efficiency of each Kt structure is obtained by data conversion for modeling. (Figure. 3b)

![]( https://static.igem.org/mediawiki/2019/c/c5/T--SYSU-CHINA--DemonsF3.png)

**Figure 3** This figure shows the quantitative and qualitative characterization of the L7Ae/L7AeO-Kturn system. **a.** Determination of inhibition efficiency of LA7e/L7AeO and K-turn at different concentrations.(Blue：L7AeO；Black：L7Ae；Green：control plasmid pcDNA). **b.** The effect of different K-turn numbers on L7Ae/L7AeO, only 2 Kt-turn can achieve greater suppression efficiency.

#### miRNA target

Based on our analysis, we designed three different miRNA targets. We inserted it into the following miRNA detection plasmid (Figure 4a) to verify its independent ability to work and detect the expression of the corresponding miRNA in different HEK293. To verify whether the system causes tandem, we constructed three different plasmid contains 3 miRNA targets independently (miR-592, The detection plasmids of miR-663b and miR885-5p) were tested under different miRNA induction. After the plasmid is transcribed into the cell, the endogenous miRNA binds to the target on the mKate mRNA, blocking the expression of mKate, which cause a change in the value of mKate/EBFP that can be read by flow cytometry. The detection plasmids were tested under different miRNA induction.

By this method, we can compare the expression of the endogenous corresponding miRNA with the Positive Control which does not carry the miRNA target. At the same time, we transferred miR-592 mimetic, miR-885-5p and miR-663b inhibitors into corresponding cells to detect the regulation of the corresponding miRNA target by miR concentration, thus verifying the rationality and feasibility of single miRNA target design.

In the flow cytometry analysis (Fig. 4b, 5), we refer to the group with the lower affected group of mKate as the positive group, and the group with the larger influence of mKate as the negative group. We can identify a group of cells that are not obvious in the negative group (hexagonal circled part) in the positive group, and there is a significant difference in the ratio of cells between the positive group and the negative group in the interval of mKate/EGFP<0.4. Thus, we believe that miRNA targets can respond to changes in miRNA concentration.

![]( https://static.igem.org/mediawiki/2019/d/db/T--SYSU-CHINA--MiRMeasurement.jpg)

**Figure 4** Flow cytometry of miRNA detection plasmids. **a.** The detection plasmid is mainly composed of two fluorescent expression systems. The target of the target miRNA target is inserted into the 3'UTR sequence of mKate. If the miRNA target can work normally, the fluorescence ratio of mKate/EBFP can be changed. **b.** In the results, the positive group has a common cell population, which shows that all three miRNA targets work properly. At the same time, the concentration of endogenous miRNA corresponding to HEK293 can be displayed.

![]( https://static.igem.org/mediawiki/2019/3/3d/T--SYSU-CHINA--mKate.png)

**Figure 5** Density analysis of positive and unique cell populations revealed that the positive group had a deviation from the negative expression of mKate in the negative group

#### miRNA sensor composite system work

We constructed a plasmid with a complete miRNA sensor and verified the effects of the entire system at different miRNA concentrations. First, we regulated the concentration of miRNAs in HEK293 through three different miRNA mimics and inhibitors. miRNA-592 mimetic to shut down L7Ae translation, miRNA-663b inhibitor and miR-885-5p inhibitor to block EGFP mRNA degradation. That is to say, when three miRNA mimics are simultaneously transferred, the entire system reaches the maximum working efficiency to output EGFP. The results showed that EGFP was slightly upregulated when either miRNA mimic/inhibitor combination was input, with the combination of miR-663b inhibitor + miR-885-5p inhibitor being the least efficient. This may be due to the fact that the targets of these two miRNAs are on the same mRNA, and the two miRNAs will have a competing effect. In addition, when three miRNA mimics/inhibitors were simultaneously input, we observed a significant difference in fluorescence brightness, which means that our miRNA sensor can work normally only under the expression of a specific miRNA. This undoubtedly verifies its specificity.

![](https://static.igem.org/mediawiki/2019/7/7e/T--SYSU-CHINA--CombinationOfDifferentMiRNA.jpg)

**Figure 6** Different miRNA combinations control the output of miRNA sensors

## Verify the response sensitivity of the system switch

### Quantitative detection of relationship between Dox concentration and fluorescence induction

The result shows that only 270ng/ml DOX can induce half of the gene expression(Figure 7), which means a low dose of DOX can achieve the desired effect without considering the side effect of over-dose.

![]( https://static.igem.org/mediawiki/2019/7/77/T--SYSU-CHINA--rtTA.jpeg)

**Figure 7** The effect of Dox concentration on induction efficiency, fitting half of the induction value

### Effect of rtTA3 controlled under different promoters on Tet-on system

We also constructed two plasmids which contains the rtTA3 controlled by different promoters. One promoter is pCMV, the other one is hEF1α. The result shows that under the regulation of pCMV, the variance of expression is smaller. While, hEF1α has lower background and higher expression in a high concentration of DOX.(Figure.8)

![](https://static.igem.org/mediawiki/2019/c/cf/T--SYSU-CHINA--rtTApromoter.gif)

**Figure 8** Different promoters control the effect of rtTA3 on Tet-on system

## Recombined and packaged the virus

We used the Adeasy system to insert our circuit into the Ad5 virus missing E1 and E3. First, we constructed the shuttle plasmid pShuttle-AdmiT in vitro to carry our circuit. Then transfer it with pAdeasy into E.coli BJ5183 through electroporation. These two plasmids were recombined in E. coli to obtain the Ad5-type adenovirus genome plasmid carrying our circuit. Figure b is a demonstration of a recombinant plasmid, 1 lane represents a successful recombinant plasmid, lane 2 represents pShuttle, and lane 3 represents a plasmid that has not been successfully recombined.

We then introduced the plasmid into HEK293 cells and packaged the virus. The image of Day3-8 is shown in the figure. It can be seen that significant CPE appeared on the third day after transfection, and a large number of suspended cells were produced on the eighth day. We harvested the virus suspension on day 8 and stored at -80 ° C.

![](https://static.igem.org/mediawiki/2019/7/7a/T--SYSU-CHINA--packvir.png)

**Figure 9** Results of packaging recombinant adenovirus. **a)** a plasmid profile of our recombinant adenovirus **b)** The supercoiled clone in Line 1 is recombinant because it migrate at positions larger than the 10-kb marker and its shuttle vector. However line 3 is not. **c-f)** Use recombinants to transfect the packaging cell line(HEK293), and the CPE showed since 72h.p.t(hours post transfection).

### Determination of virus titer

After harvesting the virus, we determined the TCID50 of the recombinant virus. The results showed that the TCID50 of the virus was 1x10-3.6/ml (Table 1). According to our modeling results, such titers can effectively kill tumors and avoid “escape” effects.

**Table 1** The result of TDIC50 assay

![](https://static.igem.org/mediawiki/2019/b/bf/T--SYSU-CHINA--Demonstrate-virus-titter.png)

### Killer: Characterized the efficacy of genes that turn on the virus to kill tumors

We verified that E1A can restore the virus's self-replication ability by transfection experiments in HeLa cells, and test the killing effect of E1A induced by different concentrations of Dox. This shows that our design in Killer is feasible, we are verifying the impact of the E1B55K, which may be displayed on our posters and presentations. We found that E1A can induce the additive killing of adenovirus in HeLa cells, while the virus lacking E1A cannot increase in cells.

It can be seen from the cell microscopic (Figure.10) examination that the number of cell deaths increases with increasing E1A concentration. The detection of cell activity on day 4 also supported this result, indicating that our killer gene is effective. (Figure.11) When Dox is 0, E1A also causes apoptosis, which may be caused by pTRE leakage expression. At the same time, since virus infection enhances cell respiration, MTS detects mitochondrial activity, and the virus group data has partial deviation.

![](https://static.igem.org/mediawiki/2019/8/8a/T--SYSU-CHINA--E1AtransfectionPicture.jpg )

**Figure 10** Microscopic examination after four days of virus infection

![](https://static.igem.org/mediawiki/2019/b/bc/T--SYSU-CHINA--E1Agraph.jpg )

**Figure 11** Detection of mitochondrial activity by MTS staining for detection of viral effects

## The system works normally after being connected in series

Based on the above data, we verified the ability of each system to work independently. Now we will test different systems in series to prove that our circuit design is feasible.

### Tet-on system regulates the initiation of miRNA sensor

We placed the miRNA sensor downstream of the pTRE promoter to detect if Tet on could work compatibly with it. miRNA-592 mimics were transported to switch off the miRNA sensor, while miRNA-885-5p inhibitor were transported to switch on the miRNA sensor. Experimental result shows that the two systems are well compatible.

![](https://static.igem.org/mediawiki/2019/4/4a/T--SYSU-CHINA--tsys.png)

**Figure 12** Expression combination of miRNA sensor and Tet on

### Tet-on system regulates the Ad5 to kill cancer cell

We co-transfected the adenoviral genome with the E1A gene located downstream of pTRE into HeLa cells. The cells were induced by different concentrations of Dox, and it was found that with the increase of Dox concentration, the cell survival of HeLa cell decreased gradually after 4 days of transfection. This shows that our adenoviral vector system, killer gene and Tet-on system are compatible.

![](https://static.igem.org/mediawiki/2019/8/8a/T--SYSU-CHINA--E1AtransfectionPicture.jpg )

**Figure 13** Tet-on system control virus to kill cancer cell

## Conclusion

The above data can prove that our design is reasonable and feasible, and through the improvement brought by modeling, this system can be predictably applied to the tumor environment in different situations for killing.

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