iGEM 2023 — Team lzu-china

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{% block title %}Sustainable Development Goals{% endblock %}
{% block lead %}Describe how you have evaluated your project ideas against one or more of the SDGs.{% endblock %}
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Notebook

[May](#l1)
[June](#l2)
[July
ages](#l3)
[August](#l4)
[September](#l5)
[October](#l6)
[Others](#l7)

# May

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Preliminary idea determination

 Search and screening of cholesterol degrading genes

 Search for a suitable promoter: oleic acid inducer was finally identified

 The design of gene combination

# June

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Gene synthesis and plasmid construction

# July

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Prepared the competent

Bacterial culture and plasmid extraction

Transformed the target gene into the chassis organism Escherichia coli Nissel 1917

Performed cell counting to detect bacterial growth status

# August

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Functional verification of IsmA and BCoAT genes, including:

Cholesterol was measured by phthalaldehyde

 SCFAs production was measured by gas chromatography

 The bacteria were lysed by sonication for BSH crude enzyme solution extraction

# September

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Qualitative detection of bile saline hydrolase

Prepared egg yolk medium and perform the detection of cholesterol degradation from food sources

Detection of BSH enzyme activity

# October

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Verified the function of oleic acid inducer by microplate reader and mRFP

Confirmed the expression of IsmA and BSH by SDS-PAGE

Determined the downstream products of IsmA conversion to cholesterol by UHPLC

Preparation and functional verification of ACA microcapsules

# Others

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Two: the experiment of the record scanning

1、Share documents online:

![](https://static.igem.wiki/teams/4788/wiki/notebook/one.png)
![](https://static.igem.wiki/teams/4788/wiki/notebook/two.png)

2. Scanning of laboratory record book:

{% for x in range(1,35)%}
![](https://static.igem.wiki/teams/4788/wiki/notebook/{{x}}.jpg)
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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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Engineering

[Overall Engineering](#l1)
[First Iteration - Chassis Engineering](#l2)
[Second Iteration - Chassis Engineering](#l3)
[Third Iteration - Chassis Engineering](#l4)
[First Iteration - Gene Function Validation Engineering](#l5)
[Second Iteration - Gene Function Validation Engineering](#l6)
[Third Iteration - Gene Function Validation Engineering](#l7)
[First Iteration - Gene Expression Verification Engineering](#l8)
[Second Iteration - Gene Expression Verification Engineering](#l9)
[First Iteration - Oleic Acid Inducer Engineering](#l10)
[Second Iteration - Oleic Acid Inducer Engineering](#l11)
[Third Iteration - Oleic Acid Inducer Engineering](#l12)

# Overall Engineering

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Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. As in any other engineering field, synthetic biologists follow iterations of the Design-Build-Test to successfully develop innovative biological systems

![](https://static.igem.wiki/teams/4788/wiki/engineering.jpg)

# First Iteration - Chassis Engineering

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## Design

Considering the need for long-term monitoring and regulation of cholesterol levels within the gastrointestinal tract, we selected Escherichia coli Nissel 1917, which naturally adheres to the intestines, as the chassis organism for the entire system. Additionally, as we require the cultivation and testing of engineered bacteria in various types of media, compatibility between the chassis organism and experimental conditions needed to be assessed.

## Build

To do this, we separately cultured bacteria that were transformed with the cholesterol-degrading gene (IsmA) in a cholesterol-based culture medium (BCM) and performed a bacterial count after 24 hours.

## Test

Results indicated that all types of engineered bacteria grew normally in the experimental culture medium.

## Learn

Based on the above findings, we proceeded to the next step, which involves detecting the target substances in the culture medium.

# Second Iteration - Chassis Engineering

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## Design

In this iteration, we plan to employ the o-phthalaldehyde (OPA) method to detect cholesterol content in the culture medium. Engineered bacteria containing the IsmA gene will be cultured in BCM for 48 hours, and a sterile control group will be established.

## Build

We prepared a cholesterol-based culture medium with a cholesterol concentration of 0.2 mg/ml and activated the engineered bacteria in LB medium. After 12 hours, the bacteria were inoculated into BCM, and the cholesterol content in the culture medium was measured at specified time intervals.

## Test

After 48 hours of culture, we extracted the culture fluid, centrifuged it, and removed the bacteria from the medium using a 30kDa filter membrane. This step was taken to eliminate interference from cholesterol naturally present in the bacteria.
Samples:
1. BCM without bacterial inoculation.
2. Engineered bacteria with four different genotypes containing the IsmA gene.

## Learn

We first dissolved cholesterol in methanol/ethanol and then added the cholesterol-containing ethanol to the BCM mother liquor to prepare the culture medium. However, due to polarity changes caused by the mixture, a significant amount of cholesterol precipitated after being added to the mother liquor. This resulted in cholesterol adsorbing to the test tube wall during the cultivation, preventing its degradation by the bacteria (which also explained the initially strange data). Therefore, we decided to reduce the cholesterol concentration to 20 micrograms per milliliter and retest the changes in cholesterol content during the engineered bacteria's degradation process.

# Third Iteration - Chassis Engineering

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## Design

In this project, we conducted a reevaluation of cholesterol levels in BCM under the influence of the IsmA gene, which garnered a positive response. Subsequently, we simulated the degradation process of engineered bacteria in natural foods. For further details, please refer to the Gene Function Verification Engineering Module. After completing this phase of testing, we anticipate incorporating capsules and a self-destruct mechanism into our engineered bacteria to ensure the smooth execution of their various functions.

## Build

We created microcapsules using sodium alginate and chitosan (also known as ACA) and collaborated with Beijing University of Chemical Technology (BUCT) to design an oxygen suicide switch. For more details, please refer to the hardware and concept validation.

## Test

We verified the viability of EcN (engineered bacteria) when encapsulated and not encapsulated after treatment with 4% bile salt solution and simulated gastric fluid (SGF). Additionally, we measured the viability of EcN with the introduction of an oxygen suicide switch under aerobic and anaerobic conditions (suicide switch data sourced from BUCT).

## Learn

The data indicates a significant increase in the viability of EcN when encapsulated with ACA, after exposure to 4% bile salt solution and SGF treatment. Furthermore, EcN introduced with the oxygen suicide switch cannot grow under aerobic conditions. In the subsequent stages, we may need to consider how to deliver the engineered bacteria to the most suitable site for their intended function.

# First Iteration - Gene Function Validation Engineering

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## Design

In this phase of the project, we commence the systematic validation of genes with distinct cholesterol-regulating functions and their corresponding roles while preliminary testing the efficacy of our detection methods.

## Build

IsmA: Employing the OPA method, we measured the cholesterol decrease before and after cultivation, using two distinct techniques:
1. Preparation of cholesterol-based cultivation medium (BCM) followed by testing.
2. Simulating high cholesterol sources in food by preparing yolk-based cultivation medium and subsequent testing.
In addition, we conducted Ultra-Performance Liquid Chromatography (UPLC) to detect other byproducts during the IsmA metabolism, thereby confirming its functional role.

BSH: Qualitative testing of the bile salt hydrolase enzyme expression by the BSH gene was conducted using MRS plates with sodium taurocholate.
Furthermore, through extraction of crude enzyme liquid from the BSH gene-engineered strains and using the indole-3-acetic acid method, we assessed the enzyme's activity based on the amount of amino acids produced by bile salt hydrolysis.

BCoAT: We employed Gas Chromatography (GC) to measure the expression of three short-chain fatty acids, including acetic, propionic, and butyric acid, as an indicator of the BCoAT gene's functionality.

## Test

The results indicate that the IsmA gene efficiently accomplished its task of reducing cholesterol levels, with more pronounced effects in the medium mimicking natural food. UPLC results further confirm the functional role of the IsmA gene.
Qualitative BSH testing and enzyme activity measurements similarly demonstrate its functionality.
Additionally, gas chromatography data suggests normal functionality of the BCoAT gene.

## Learn

In this round of functional validation engineering, the excellent data results underscore the effectiveness of our testing methods, signifying their suitability for continued use in subsequent project iterations. However, it is essential to note that in this round of functional testing, we exclusively examined engineered strains carrying single genes. As per our initial design, we need to concatenate different genes and systematically test their cholesterol-reducing efficacy, ultimately leading to the optimal cholesterol degradation solution, which will be explored in the forthcoming project phases.

# Second Iteration - Gene Function Validation Engineering

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## Design

In this phase of the project, we have concatenated IsmA, BSH, and BCoAT genes in pairs to obtain two gene combinations: IsmA-BSH and IsmA-BCoAT. We applied the same methods to assess their functionalities.

## Build

The methods used are consistent with the previous iteration, involving OPA, UPLC testing, qualitative BSH testing, enzyme activity assessment, and gas chromatography for the evaluation of individual gene functionality within different gene combinations.

## Test

Data results indicate that when two genes are concatenated, the ability of IsmA to directly degrade cholesterol shows improvement. In qualitative observations and BSH enzyme activity testing, bile salt hydrolase activity appears to increase. However, in the testing of short-chain fatty acids (SCFAs) expression, the production decreases relative to when the BCoAT gene exists independently.

## Learn

In this round of testing, we unexpectedly discovered differences in gene functionality when different genes are combined compared to their performance when present individually. This suggests the existence of potential unknown interactions between the three genes. Whether this hypothesis holds true requires extensive experimentation. However, this intriguing finding sparks substantial interest in the results of assembling all three genes together in the future.

# Third Iteration - Gene Function Validation Engineering

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## Design

In this phase, our focus has shifted to engineered strains simultaneously carrying all three genes.

## Build

Our testing methods remained consistent with the previous two iterations.

## Test

The results were both expected and surprising. The IsmA gene continues to exhibit increased values in the cholesterol degradation process, with an absolute reduction in cholesterol levels in the cultivation medium. Qualitative BSH testing forms larger precipitate rings, and total enzyme activity increases, although unit enzyme activity seems to decrease. Furthermore, SCFAs expression levels remain lower than when each gene is present individually and even lower compared to the two-gene concatenation.

## Learn

The results from this project phase seem to further support the findings from the second iteration. However, due to the absence of significant or only weakly significant differences in some data sets, continued experimentation and method refinement are required to verify these conclusions. In future experiments, we will persist in examining this hypothesis.

# First Iteration - Gene Expression Verification Engineering

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## Design

To validate gene expression and align with the gene function verification engineering iteration, we initially focused on the BSH gene. The enzyme activity assay for BSH served as a validation for BSH protein secretion.

## Build

Similar to the gene function verification engineering, we extracted crude enzyme solutions from different strains of engineered bacteria carrying the BSH gene. We measured the enzyme's total activity by quantifying the amount of amino acids produced through BSH hydrolysis of bile salts using the 3-oxoketone assay. Combining this with protein expression measurements allowed us to obtain the average enzyme activity.

## Test

The presentation of enzyme activity data confirmed the expression of the BSH protein.

## Learn

While we demonstrated BSH protein expression, our validation remained function-based. We need to explore methods for protein-level validation of gene expression.

# Second Iteration - Gene Expression Verification Engineering

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## Design

We chose SDS-PAGE gel electrophoresis to validate the expression of BSH and IsmA genes at the protein level.

## Build

We cultured the target strains in LB for 24 hours, obtained cell lysate using an ultrasonicator, centrifuged to obtain cell-free extracts, and subsequently used SDS-PAGE gel electrophoresis to separate proteins.

## Test

We successfully separated the target proteins in SDS-PAGE electrophoresis, validating gene expression.

## Learn

While we separated proteins through SDS-PAGE electrophoresis, further protein validation is necessary to obtain clearer results. In subsequent iterations, we should consider Western blotting (WB) for protein separation.

# First Iteration - Oleic Acid Inducer Engineering

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## Design

To characterize the functionality of the oleic acid inducer, we appended the mRFP gene to the inducer after oleic acid induction for fluorescence characterization, thereby verifying the inducer's function.

## Build

We conducted oleic acid induction experiments using engineered bacteria carrying the oleic acid inducer-mRFP gene. The level of inducer expression was characterized using a spectrophotometer.

## Test

We successfully completed the oleic acid induction, demonstrating that oleic acid effectively induced the inducer's expression.

## Learn

While we validated the basic functionality of the oleic acid inducer, given our target environment in the anaerobic gut, we need to consider functional testing under anaerobic conditions.

# Second Iteration - Oleic Acid Inducer Engineering

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## Design

To test if the oleic acid inducer functions properly under anaerobic conditions, we cultured the oleic acid inducer-mRFP engineered bacteria in an anaerobic environment.

## Build

We achieved anaerobic cultivation of the target engineered bacteria using anaerobic bags. Post-cultivation, we characterized the inducer's expression using a spectrophotometer.

## Test

We successfully conducted anaerobic cultivation of the oleic acid inducer strain, which responded well to oleic acid induction, displaying even more significant expression compared to aerobic conditions.

## Learn

In this iteration, we verified the basic functionality of the oleic acid inducer under high oleic acid induction. To further confirm oleic acid as the triggering condition and study the potential impact of different oleic acid induction concentrations on gene expression mediated by the inducer, we need to explore gradient inductions.

# Third Iteration - Oleic Acid Inducer Engineering

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## Design

To test gradient induction by the oleic acid inducer, we used gradient oleic acid culture media to induce the expression of the oleic acid inducer-mRFP.

## Build

We prepared gradient oleic acid culture media and cultivated strains carrying the oleic acid inducer-mRFP under aerobic and anaerobic conditions. Post-cultivation, we characterized the inducer's expression using a spectrophotometer.

## Test

We successfully detected the oleic acid inducer's response to gradient oleic acid, confirming its ability to respond to varying oleic acid concentrations.

## Learn

In this engineering cycle, the oleic acid inducer exhibited higher fluorescence protein expression under high oleic acid induction, confirming its responsiveness. In future experiments, we aim to adjust the operator's quantity to widen the oleic acid induction threshold range, aligning with our initial goals for a more intelligent and customized system. Additionally, we plan to connect the oleic acid inducer to the optimal gene genotype for cholesterol degradation to evaluate and obtain optimal gene expression data under the effect of the inducer.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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{% block title %}Results{% endblock %}
{% 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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