iGEM 2024 — Team gslz-a


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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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## Overall Engineering

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Synthetic biology centers on the design and construction of new biological parts, biological systems, and even completely synthetic organisms to perform specific functions. In this project, we hope to achieve barrier clearance and immune enhancement of the OSCC tumor microenvironment through synthetic biology.

## 1.1. First Iteration - Chassis Engineering

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* **Design**

  In this iteration, we mainly explored the compatibility between chassis bacteria and experimental conditions.
* **Build**

  In the design, we used L.B. liquid medium and nutrient agar plates to culture the chassis bacteria E. coli Nissle 1917 and the engineered bacteria containing the plasmid. We will verify whether the chassis bacteria and engineered bacteria can grow in the same state on L.B. medium using the dilution plating method. Additionally, we will test the biological activity of both bacteria using a bacterial viability detection kit to confirm equal activity under the same experimental conditions. Furthermore, since we need to perform time series measurements throughout the project, we will plot the growth curves of both chassis and engineered bacteria under identical conditions to further assess their states.
* **Test**

  The results indicated that there were no significant differences in colony counts, bacterial activity, and growth curves between the chassis bacteria and the engineered bacteria in L.B. medium. This suggests that the experimental conditions we established do not interfere with the experimental outcomes.
* **Study**

  Based on the findings mentioned above, we established the experimental conditions and proceeded with the next iteration to validate the expression of the target gene.

## 2.1. First Iteration - Gene Expression Verification Engineering

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* **Design**

  In this iteration, we primarily aimed to verify the successful integration and expression of the recombinant plasmid in the chassis bacteria, which is crucial for the subsequent phases of our research.
* **Build**

  Due to the presence of a kanamycin resistance fragment in the recombinant plasmid, we chose to directly plate the chassis bacteria and engineered bacteria on commercial kanamycin L.B. agar plates (antibiotic concentration > 100 minimum inhibitory concentration) to assess their differences. Additionally, we extracted the plasmid from the engineered bacteria and performed restriction enzyme digestion, followed by agarose gel electrophoresis to confirm the presence of the recombinant plasmid.
* **Test**

  The results indicated that the engineered bacteria were able to grow and form normal colonies on the kanamycin agar plates, while the chassis bacteria did not show any colony formation. This suggests that the recombinant plasmid was successfully expressed in the engineered bacteria. The agarose gel electrophoresis results showed that the plasmid from the engineered bacteria was cut into two fragments as expected.
* **Study**

  Based on the findings mentioned above, we have demonstrated that the recombinant plasmid has been successfully transferred into the engineered bacteria and expressed. In the upcoming experiments, we will further validate the expression of the target protein.

## 2.2. Second Iteration - Gene Expression Verification Engineering

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* **Design**

  In this iteration, we verified the expression of hyaluronidase in the secreted and lysate fractions of the engineered bacteria to assess protein expression and release.
* **Build**

  We collected engineered and chassis bacterial suspensions cultured between the logarithmic and stationary phases, dividing them into two parts. For the detection of bacterial secretions, we used high-speed centrifugation to remove the bacteria from the suspensions and performed an ELISA to assess the hyaluronidase concentration in the supernatant according to the His ELISA kit instructions. For the bacterial suspensions, we performed sonication on ice, followed by protein concentration measurement using the BCA assay, and detected the expression of hyaluronidase-His using SDS-PAGE and Western blotting.

  Additionally, we measured the hyaluronidase concentration in the bacterial lysate using the same ELISA method as in the previous steps to assess the release rate of the target protein.
* **Test**

  The results of the Western blot indicate that the molecular weight of the recombinant hyaluronidase is between 14-15 kDa, which is consistent with expectations. The concentration of hyaluronidase in the supernatant is approximately 51.25% of that in the lysate.
* **Study**

  Based on the findings above, we have confirmed the successful expression and secretion of hyaluronidase in the engineered bacteria.

## 2.3. Third Iteration - Gene Expression Verification Engineering

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* **Design**

  In this iteration, we verified the expression of the target product, Flab, in the engineered bacteria.
* **Build**

  We collected bacterial suspensions cultured for 12 hours, between the logarithmic and stationary phases, and performed sonication on ice, followed by protein quantification using the BCA assay. Subsequently, we assessed the expression of the target protein Flab using SDS-PAGE and Western blotting with Flag tag antibodies.
* **Test**

  The Western blot results indicate that Flab is expressed in the engineered bacteria, with a molecular weight between 13-15 kDa, consistent with predictions.
* **Study**

  Based on the findings above, we have demonstrated the successful expression of the target protein and will proceed to validate its functionality in the subsequent experiments.

## 3.1. First Iteration - Gene Function Validation Engineering

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* **Design**

  In this iteration, we verified the ability of hyaluronidase to degrade extracellular matrix analogs and measured the enzyme activity using a hyaluronidase activity detection kit to assess the biological function of the target protein.
* **Build**

  We simulated the extracellular matrix in vitro using photocrosslinked hyaluronic acid methacrylate (HAMA) hydrogels and continuously incubated them with bacterial culture supernatant for 72 hours. Every 12 hours, we removed the hydrogels, freeze-dried them, and measured the dry weight changes over time. Subsequently, we measured the enzyme activity in the bacterial culture supernatant using a hyaluronidase activity detection kit and a spectrophotometer.
* **Test**

  The results indicate that the degradation rate of the extracellular matrix-like hydrogel reached 56.7% within 48 hours, accompanied by morphological disintegration. Enzymatic activity tests show that the hyaluronidase activity in the engineered bacterial supernatant is comparable to that of the commercial product (50 µg/ml).
* **Study**

  Based on the above findings, we confirmed that the engineered bacteria can produce and secrete hyaluronidase, which is sufficient to degrade the extracellular matrix and achieve our intended functionality.

## 3.1. Second Iteration - Gene Function Validation Engineering

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* **Design**

  In this iteration, we compared the differences in immune system activation between the chassis bacteria and the engineered bacteria to infer whether Flab enhanced immunogenicity.
* **Build**

  We cultured mouse macrophages (RAW264.7) purchased from a commercial company in our dedicated cell culture facility and co-cultured them with a logarithmic phase bacterial dilution. Subsequently, we used BCA protein quantification and Western blot methods to verify the expression of the macrophage polarization marker protein iNOS, in order to infer the activation status of the macrophages.
* **Test**

  The results indicate that the iNOS expression in macrophages incubated with the engineered bacteria was upregulated, suggesting that Flab enhances the immunogenicity of the engineered bacteria.
* **Study**

  Based on the above research, we confirmed the function of the recombinant protein Flab. However, upon reviewing the entire experiment, we found gaps in how the engineered bacteria are delivered to tumor tissue and how normal tissues are protected. This will be more thoroughly designed in future studies.

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