iGEM 2023 — Team gec-guangzhou

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[Week 1 Experimental Preparation](#a1)

[Week 2: Plasmid carrying strains were activated, and plasmid templates were extracted to begin constructing the plasmid.](#a2)

[Week 3 Gel electrophoresis, gel recovery, double enzyme digestion, recovery](#a3)

[Week 4: Preparation of Competent Cells](#a4)

[Week 5: Ligation and Transformation](#a5)

[Week 6: Verification of Plasmid Construction and Expansion Cultivation](#a6)

[Week 7: Measurement of CDase Activity](#a7)

[Week 8: Effects of 5-FC and pT7-CD on Cell Viability](#a8)

[Week 9: HlpA promotes the adhesion of engineered bacteria to colorectal cancer cells.](#a9)

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Week 1 Experimental Preparation

Experiment Plan: Prepare culture media and experimental consumablesExperimental Procedure:1. Sterilization: It is recommended to wrap Eppendorf tubes, PCR tubes, and centrifuge tubes with gauze. Load large, medium, and small-sized tips separately, and wrap the shells with parchment paper. To fix the tip box, you can use ropes or rubber bands for bundling. At the same time, LB media that have been prepared should be aliquoted into 20 test tubes, and 2 bottles of 50mL LB liquid media need to be prepared. To ensure sterility, place the above items in a high-pressure autoclave for sterilization at 121°C for 15 minutes.

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Week 2: Plasmid carrying strains were activated, and plasmid templates were extracted to begin constructing the plasmid.

Experimental plan: Plasmid extraction, extraction of plasmid templates, PCR amplification of target fragments. Experimental procedure: 1. Activate strains: Including 1 tube of Escherichia coli DH5α/empty vector, 1 tube of DH5α/carrying CD gene, and 1 tube of Escherichia coli DH5α/inp-hlpA. Open the laminar flow hood, ventilate, place sterilized LB tubes in the hood, light an alcohol lamp, burn the tube mouth and tube cap. Add 100 μL of preserved bacterial solution to each tube, add 2.5 μL of Amp antibiotic stock solution, and burn the tube mouth and tube cap again. Tighten the tube cap, bundle the tubes, and incubate overnight on a shaker (150 rpm, 37℃). 2. Plasmid extraction: Extract the three types of plasmids (1 tube of Escherichia coli DH5α/empty vector, 1 tube of DH5α/carrying CD gene, and 1 tube of Escherichia coli DH5α/INP-hlpA) according to the instructions of the plasmid extraction kit.

![](https://static.igem.wiki/teams/5001/wiki/note/img2.jpg)

2. Dilute the primersCentrifuge the powdered primers at 12000rpm for 1 minute, and dilute the primers to the appropriate concentration by adding the corresponding amount of distilled water.

![](https://static.igem.wiki/teams/5001/wiki/note/img3.jpg)

3. PCR reaction:(1) Amplify the target gene empty vector, CD, and DNA fragments using high-fidelity enzyme Pfu. The reaction system is as follows:Pfu 0.5 μL10x Pfu buffer 5 μLPrimer 1 1 μLPrimer 2 1 μLTemplate (plasmid extracted in the previous step): 1 μLdNTP Mixture 25 μLSterile water 20 μL(2) Perform PCR amplification program:98℃, 5 minThe following 3 steps are repeated for 30 cycles:98℃ 10s55℃ 5s72℃ 1 min/kb72℃ 8 min

![](https://static.igem.wiki/teams/5001/wiki/note/img4.jpg)

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Week 3 Gel electrophoresis, gel recovery, double enzyme digestion, recovery

Experimental plan: Gel electrophoresis, gel recovery, double enzyme digestion, recoveryExperimental procedure:1. Gel electrophoresis: Add the above PCR product to the prepared agarose gel, add 5 μL DL 5000 DNA marker, and perform electrophoresis (120V, 20 min). After electrophoresis, observe using a gel imaging system, cut the desired DNA gel, and place it in a 2 mL centrifuge tube for labeling.

![](https://static.igem.wiki/teams/5001/wiki/note/img5.jpg)

2. Gel recovery: Perform gel recovery on the PCR product DNA gel according to the instructions of the gel recovery kit. Add 500 μL of equilibration buffer BL to the adsorption column CA2, centrifuge for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Cut the single desired DNA band from the agarose gel and place it in a clean centrifuge tube. Add an equal volume of solution PN to the gel piece, incubate at 50 degrees Celsius until the gel is completely dissolved. Transfer the obtained solution to another adsorption column CA2, let it stand at room temperature for 2 min, and centrifuge for 1 min. Add 600 μL of washing solution PW to the adsorption column CA2 (check if ethanol has been added before use), centrifuge for 1 min, repeat the washing process twice, and air dry. Add an appropriate amount of elution buffer EB to the center of the adsorption membrane, let it sit at room temperature for 2 min. Centrifuge for 2 min and collect the DNA solution. Measure the concentration of the recovered DNA using a Nanodrop.

![](https://static.igem.wiki/teams/5001/wiki/note/img6.jpg)

3. Double Digestion: Perform double digestion on the extracted plasmid pET23b and amplified target DNA fragment using EcoRI and XhoI. Prepare a 20 μL reaction with a final volume of 20 μL. The components of the 20 μL digestion system are as follows:DNA - 1 μgEcoRI - 1 μLXhoI - 1 μL10x Y buffer - 4 μLH2O (add water) up to 20 μLIncubate the digestion reaction at 37°C for 4 hours.

![](https://static.igem.wiki/teams/5001/wiki/note/img7.jpg)

4. Agarose Gel Electrophoresis: Add the digested products to an agarose gel and add 5 μL of DL 5000 DNA marker. Perform electrophoresis at 120V for 20 minutes. After the electrophoresis is complete, use a gel imaging system for observation. Cut out the desired DNA fragment from the gel and transfer it to a 2 mL centrifuge tube for labeling. The product extraction is successful but the quantity is low. Perform a second gel purification.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Week 4: Preparation of Competent Cells

Experimental Plan: Activate E. coli DH5α and Rosetta, prepare competent cells.Experimental Procedure:1. Activation of cell strains: including Escherichia coli DH5α and Rosetta. Open the laminar flow hood, ensure proper ventilation, place sterilized LB tubes in the hood, ignite an alcohol burner, flame the tube openings and caps, add 100 μL of preserved bacterial stock solution to each tube, flame the tube openings and caps again. Close the tubes tightly, bundle them together, and incubate them overnight on a shaker (220 rpm, 37℃).Experimental Procedure:2. Preparation of Competent Cells:(1) Open the laminar flow hood and place 1 mL, 200 μL, 5 mL, 2 mL, and 30 mL centrifuge tubes, as well as 2 x 50 mL LB culture media inside. Perform UV irradiation. Pre-chill 0.1 M CaCl2 and 15% 0.1 M CaCl2 solution in a refrigerator at 4℃.(2) Prepare DH5α and Rosetta competent cells using the following steps: Place the pre-activated DH5α and Rosetta bacteria in a pre-cooled centrifuge at 4℃, centrifuge at 5000 g for 10 minutes, discard the supernatant, add 800 μL of 0.1 M CaCl2 solution, and incubate on ice for 40 minutes. Centrifuge again (4000 g, 10 min), discard the supernatant, add 100 μL of 15% glycerol in 0.1 M CaCl2 to each tube, resuspend the bacteria. Transfer the competent cells to 2 mL centrifuge tubes on ice, 100 μL per tube. Label and store the competent cells at -80℃.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Week 5: Ligation and Transformation

Experimental Plan: Prepare solid agar plates with antibiotics, perform ligation reaction, transform the ligated product.Experimental Procedure:1. Prepare antibiotic plates: Prepare LB agar medium (add 4.5 g agarose to 300 mL LB liquid medium), autoclave at 121℃ under high pressure for 15 minutes. Allow it to cool to approximately 60℃, add 150 μL of 100 mg/mL Ampicillin antibiotic, mix well, and pour into disposable petri dishes (enough for 10-15 plates).

![](https://static.igem.wiki/teams/5001/wiki/note/img8.jpg)

T4 DNA ligase 1 μL10x ligation buffer 2 μLLinear plasmid 1 μLInsert DNA 6 μLPerform the ligation reaction at 16℃ in a PCR machine for 1 hour. Obtain pET23b-insert, and store the ligated product at -20℃.3. Transformation of the ligated product: Add 10 μL of the ligation product mentioned above to 100 μL of competent cells, incubate on ice for 30 minutes, heat shock at 42℃ for 60 seconds, cool on ice for 5 minutes, and then add 1 mL of LB liquid medium. Incubate for 1 hour for recovery (37℃, 150 rpm). In the clean bench, take 100 μL of the recovery solution and spread it on an Ampicillin agar plate using a sterile inoculation loop.

![](https://static.igem.wiki/teams/5001/wiki/note/img9.jpg)

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Week 6: Verification of Plasmid Construction and Expansion Cultivation

Experimental Plan:1. Colony PCR verification: Label 6 colonies on each plate. Take half of the colonies as templates and perform colony PCR using Taq enzyme. Primers TF and TR are used. Follow the instructions for reaction system and program. Prepare a 50 mL agarose gel and perform electrophoresis using DL 2000 DNA marker. Presence of correctly sized bands indicates successful plasmid construction.2. Expansion cultivation: Pick the colonies that were verified correctly and add them to 5 mL of LB liquid medium. Add 2.5 μL of Ampicillin and perform expansion cultivation.3. Plasmid extraction: Extract the two types of plasmids according to the instructions of the plasmid extraction kit. After centrifugation of the bacterial culture for 1 minute, collect the pellet. Add p1 resuspension, invert and mix with p2, invert and mix with p3, and then centrifuge for 10 minutes. Add 500 μL of BL reagent to the column, discard the flow-through, add the supernatant obtained from previous centrifugation, wash the column twice with pw wash solution, each time centrifuging for 1 minute. Add 50 μL of EB solution to the membrane of the column, centrifuge for 2 minutes. Measure the DNA concentration of the recovered DNA using Nanodrop and store the DNA at -20℃. This step was successful for most people in extracting the plasmids.4. Transformation: Transform the verified plasmid 1 into expression type Escherichia coli.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Week 7: Measurement of CDase Activity

Culture the seed in LB medium. Take 10 mL of bacterial culture, centrifuge to remove the supernatant, resuspend the cell pellet in 20 mM Tris HCl (pH 7.0), and collect the crude enzyme solution after ice pre-sonication treatment. Measure the protein concentration using the Bradford method. Incubate the crude enzyme solution at 37℃ with 5 mg/mL 5-FC (F123460, Aladdin) for 12 hours. Extract 5-FU with methanol, dry the extract using a vacuum centrifuge, and resuspend the sample with 50 μL of methanol. Take 10 μL aliquots of the samples and add 190 μL of 0.1 M HCl. Prepare a standard curve for 5-FU using a volumetric flask and measure the 5-FU content in the samples at 266 nm using a spectrophotometer.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Week 8: Effects of 5-FC and pT7-CD on Cell Viability

1. Effects of 5-FC on cell viability: Culture CT26 cells in a 24-well plate. When the cells are confluent, add different concentrations of 5-FC (F123460, Aladdin) or PBS (control) and incubate at 37℃ for 72 hours. Measure cell viability using a CCK8 analysis kit (beyotime, C0037). The kit instructions are provided separately.2. Effects of pT7-CD on cell viability: Culture the recombinant bacteria Rosetta in LB medium. After 12 hours, collect the cells and adjust OD600 to 1 with PBS. Add 10 mM 5-FC and incubate at 37℃ for 12 hours. Centrifuge the cells.

![](https://static.igem.wiki/teams/5001/wiki/note/img10.jpg)

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Week 9: HlpA promotes the adhesion of engineered bacteria to colorectal cancer cells.

1. Thaw the cells stored in liquid nitrogen at -196°C quickly to 37°C. Inoculate the cells into a 25 cm2 culture flask with 5 mL of complete DMEM medium (referred to as "complete medium") containing 10% fetal bovine serum (Gibco, Thermor Fisher) and 1% gentamicin. Resuscitate the CT26 mouse colorectal cancer cell line and the RKO human colorectal cancer cell line in a cell culture incubator at 37°C and 5% CO2. Once the cells form a monolayer, remove the DMEM medium. Digest the cells with 1 mL of trypsin for 1 minute, followed by 1 mL of complete medium to stop the digestion. Collect the cells and inoculate them into a 24-well plate. Use a microscope to observe the growth status of the cells, ensuring that they reach approximately 70%-80% confluency in the wells.Activate the recombinant engineered bacteria by inoculating them into LB medium. Adjust the OD600 to 0.6 on the following day. Inoculate 100 μL of the engineered bacteria into the wells of the 24-well plate (at this point, the wells are fully covered with cells). Incubate for 3 hours, then remove the supernatant and wash the wells three times with PBS.Add Triton X-100 (1% volume/volume ratio) to the wells and incubate at 28°C for 10 minutes to lyse the CT26 cells. Dilute the harvested cell lysate 10,000 times by gradient dilution, and spread it onto LB agar plates containing 50 mg/L ampicillin. Incubate for 12 hours, and then count the colony-forming units (CFUs) of the adherent bacteria on the next day. Repeat this assay three times to ensure reproducibility of the measurement.

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[Overview](#a1)

[Cycle 1 Evaluation of the effect of 5-FU on cell viability and constructe pT7-CD engineered bacterial strain.](#a2)

[Design](#b1)

[Build](#b2)

[Test](#b3)

[Learn](#b4)

[Cycle 2 Enhance engineered bacterial adhesion to colorectal cancer cells](#a3)

[Design](#b5)

[Build](#b6)

[Test](#b7)

[Learn](#b8)

[Cycle 3 Suicide System - Temperature-Controlled Promoter Tcl42-pR-pL and mazF](#a4)

[Design](#b9)

[Build](#b10)

[Test](#b11)

[Conclusion](#a5)

[References](#a6)

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Overview

![](https://static.igem.wiki/teams/5001/wiki/engi/img1.png)

Figure 1 Illustration of reaction principle.

Synthetic biology primarily involves the rational design and engineering of biological components, devices, or systems. Similar to other engineering fields, synthetic biologists follow a design-build-test iterative process to successfully develop innovative biological systems.
Through genetic engineering technology, our goal is to treat colorectal cancer. Here, we will provide a detailed description of the iterative process involved in our strain engineering. First, we conducted tests on CD enzymes, which can convert 5-FC into 5-FU, making it a key factor in cancer treatment. Next, we tested the engineered bacteria that overexpress INP-HlpA for their adhesive effects on colorectal cancer cells. Finally, we used a temperature-controlled promoter to regulate the expression of the mazF cleavage gene to ensure biosecurity. All of these genetic engineering processes were validated in the laboratory strain E. coli Rosetta. In the future, we hope to integrate these components into E. coli Nissle 1917. We chose Escherichia coli Nissle 1917 (EcN) as our engineered microbial host because it possesses non-pathogenic characteristics and an excellent genetic background. This provides a solid foundation for our applications. Furthermore, EcN has a stable genetic background, making it easy to manipulate genetically.

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Cycle 1 Evaluation of the effect of 5-FU on cell viability and constructe pT7-CD engineered bacterial strain.

Design

Since the chemotherapy drug 5-FU is harmful to other normal cells in the human body, while 5-FC is harmless, we have decided to convert 5-FC into 5-FU in the intestines. Therefore, through genetic engineering technology, we have enabled Nissle1917 to express CDase. In the intestines, cytosine deaminase (CD) converts a non-toxic prodrug (Theys, Jan, et al. ) , 5-fluorocytosine (5-FC), into a toxic chemotherapy drug, 5-fluorouracil (5-FU), reducing the harm of 5-FU to the human body.

Build

We cloned the CD gene (codA) into the pET23b plasmid, using the T7 promoter and B0015 terminator as gene circuit control system. We then transferred the constructed plasmid into E. coli Rosetta (host cells).

![](https://static.igem.wiki/teams/5001/wiki/engi/img2.png)

Figure 2 Design of gene circuit for CDase overexpression.

Test

![](https://static.igem.wiki/teams/5001/wiki/engi/img3.png)

Figure 3 The experimental results related to CDase.

**Test 1: The impact of 5-FU on cell activity**
The impact of 5-FU on cell activity was assessed in CT26 cells cultivated in a 24-well plate. Following complete cell seeding, varying concentrations of 5-FU (IF0170, Solarbio) or PBS (control) were introduced, and the cells were incubated at 37°C for 36 hours. Utilizing the CCK8 assay kit (Beyotime, C0037), we determined cell viability. It was observed that the survival rate of CT26 cells gradually diminished with higher concentrations of 5-FU (Figure 3A).
**Test 2: The impact of 5-FC on cell activity**
Similarly, we investigated the effects of 5-FC on cell activity in mouse colorectal cancer cell line CT26 cultured in a 24-well plate. After full cell seeding, diverse concentrations of 5-FC (F123460, Aladdin) or PBS (control) were added, and the cells were incubated at 37°C for 72 hours. Cell viability was assessed using the CCK8 assay kit (Beyotime, C0037). Notably, there was no significant alteration in the survival capability of CT26 cells with increasing concentrations of 5-FC (Figure 3B).
**Test 3: Measuring the CD enzyme activity of the pT3-CD strain**
Subsequently, the CD enzyme activity of pT3-CD strain was determined. The engineering strain pT7-CD was cultured in LB medium. 10 mL of bacterial culture was taken, centrifuged to discard the supernatant, resuspended in 20 mM TrisHCl (pH 7.0) to collect cell pellets, and then subjected to ice pre-sonication treatment to collect crude enzyme solution. The protein concentration was determined using the Bradford method. The crude enzyme solution (5 mg/mL) was incubated with 15 mM 5-FC (F123460, Aladdin) for 12 hours at 37°C. Afterwards, 5-FU was extracted using methanol, the extract was dried using a vacuum centrifuge, and the sample was suspended in 50 μL methanol. 10 μL of the sample was added to 190 μL of 0.1 M HCl. A standard curve for 5-FU (SF8400, Solarbio) was prepared using a volumetric flask, and the sample was detected at 266 nm using a spectrophotometer. Figure C shows the activity of the CDase of the engineering strain pT7-CD (Figure 3C).
**Test 4:**
We further tested the effect of engineered probiotics on the activity of CT26 cells. The engineered EcN were cultured in LB medium, and after 12 hours, the bacterial cells were collected. The OD600 was adjusted to 1 with PBS, and 10 mM 5-FC was added. The cells were incubated at 37°C for 12 hours. The supernatant was collected as a test sample and added to CT26 cells cultured in a 24-well plate. After incubation for 72 hours, cell viability was determined using the CCK8 assay kit (beyotime, C0037). It was found that the engineering strain pT7-CD significantly increased the production of 5-FU and decreased the survival ability of CT26 cells compared to the wild-type strain, as shown in Figure 3D.

Learn

By conducting interviews with experts, we gathered valuable insights regarding the large intestine's length, typically spanning between 2 to 3 meters. It came to our attention that the initial generation of engineered strains faced challenges in accurately homing in on tumor regions. Following comprehensive team deliberations, we embarked on the development of a targeting module specifically designed to secure the engineered strains onto the surface of cancer cells. This strategic innovation is geared towards enhancing the precision and overall effectiveness of our treatment approach.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Cycle 2 Enhance engineered bacterial adhesion to colorectal cancer cells

Design

We have decided to design/construct the INP-HIpA protein. Ice nucleation protein (INP) is a commonly used protein carrier in cell surface display technology (Dou, Jian-lin, et al.). It has a large region located on the outside of the cell, which can be used for the display of other proteins. We will use this protein to display the HIpA protein, which is a histone-like protein derived from Streptococcus and can bind to heparan sulfate proteoglycans (HSPG) on the surface of tumors (Boleij, Annemarie, et al.).

![](https://static.igem.wiki/teams/5001/wiki/engi/img4.png)

Figure 4 Illustration of reaction principle.

Build

We used INP as the basis for fusing the hlpA gene to get the INP-HlpA protein. We used promoter pT7 and terminator B0015 as a gene circuit control system to express INP-HlpA and transform the recombinant plasmid based on pET23b into E. coli Rosetta.

![](https://static.igem.wiki/teams/5001/wiki/engi/img5.png)

Figure 5 Design of the INP-HlpA overepression system.

Test

![](https://static.igem.wiki/teams/5001/wiki/engi/img6.png)

Figure 6 The experimental results related to INP-HlpA.

Under the same conditions, we set up assessments for E.coli Rosetta and engineered bacteria E.coli Rosetta/pT7-HlpA. We put the bacteria together with two types of colorectal cancer cells (RKO and CT26) to observe the number of bacteria adhere the cancer cells.Quantitatively, our results showed that E.coli Rosetta without pT7-HlpA have about 42\*10^5 CFU counts well of RKO cells and about 39\*10^5 CFU counts well of CT26. In comparison, E.coli Rosetta with pT7-HlpA have about 82\*10^5 CFU counts well of RKO cells lysate and about 82\*10^5 CFU counts well of CT26 cells lysate. By calculation, fusion protein pT7-HlpA enhance the ability of E.coli to adhere to RKO cells by 195% and CT26 by 210%.

Learn

After improvement, the engineered strains are now capable of providing more effective treatment for colorectal cancer.However, considering the biosafety issue, we may need to introduce a suicide system to lyse the bacterial strains after functional expression.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Cycle 3 Suicide System - Temperature-Controlled Promoter Tcl42-pR-pL and mazF

Design

The basal transcription activity of the gene can be regulated within a specific temperature range by using a temperature-controlled promoter. We chose the pTcl42 promoter because its ideal activation temperature threshold is 42°C, and it has low expression levels at 37°C (Wu, Ming-Ru, et al.). MazF is a widely studied toxin-antitoxin (TA) system in Escherichia coli, and its mechanism of action is well defined (Tripathi, Arti, et al.). MazF can recognize ACA sequences and hydrolyze the phosphodiester bond at the first A position at either the 5' or 3' end, causing ribosome release from cleaved mRNA and preventing protein synthesis. Subsequently, improperly encoded polypeptides are released and degraded by intracellular proteases, leading to cell death. Therefore, we plan to use the temperature-controlled promoter TC1-42 and mazF to induce bacterial lysis.

Build

In order to achieve temperature-induced self-lysis in the engineered strain, the pTcl42 promoter was placed upstream of the bacterial lysis gene mazF. Subsequently, the Tcl42 promoter and the mazF gene were cloned together into the pSB1A3 plasmid.

Test

The successfully constructed plasmid was transformed into E. coli DH5a bacteria using heat shock method, and recombinant bacteria were screened on LB agar plates containing 100 μg/mL ampicillin. To evaluate the expression of the mazF gene at different temperatures, the transformed bacteria were cultured for 12 hours at 37°C and 42°C, and their OD600 values were measured using a spectrophotometer. Wild-type DH5a and DH5a carrying only the pTcl42 of pSB1A3 plasmid were used as controls. All experiments were performed in triplicate to ensure the reliability of the results. The results are shown in Figure 8A.
To evaluate the time dependence of the bacterial lysis system driven by the pTcl42 promoter at 42°C, a time-course test was performed. The engineered bacteria were added to a 96-well plate and cultured in a shaking incubator at 42°C. Every 2 hours, the OD600 values were measured using a microplate reader to assess bacterial growth. The results are shown in Figure 8B. All experiments were performed in triplicate to ensure the reliability of the results.
The experimental results showed that the temperature-inducible promoter was induced at 42°C, causing almost complete lysis of the bacteria within 12 hours. In the future, we plan to modify common suppositories to have a heating function. When it is necessary to completely kill the engineered bacteria, the heating function of the suppository will be activated to initiate the bacterial suicide system.

![](https://static.igem.wiki/teams/5001/wiki/engi/img7.png)

Figure 7 Gel electrophoresis of Tcl42-pR-pL and mazF.

![](https://static.igem.wiki/teams/5001/wiki/engi/img8.png)

Figure 8 The experimental results related to pTcl42-mazF.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

Conclusion

After three rounds of iterative design, the engineered strain has been able to effectively address our conceptual experiments.

![](https://static.igem.wiki/teams/5001/wiki/pro2.png)

References

1. Boleij, Annemarie, et al. "Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells." Infection and immunity 77.12 (2009): 5519-5527.2. Dou, Jian-lin, et al. "Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein." Virologica Sinica 26 (2011): 409-417.3. Theys, Jan, et al. "Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum." Cancer Gene Therapy 8.4 (2001): 294-297.4. Wu, Ming-Ru, et al. "A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS)." Nature communications 10.1 (2019): 2880.5. Tripathi, Arti, et al. "MazF-induced growth inhibition and persister generation in Escherichia coli." Journal of Biological chemistry 289.7 (2014): 4191-4205.

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{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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