iGEM 2023 — Team squirrel-guangzhou

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

# Engineering Success

## Design

Our research aims to heterologously express steviol glycosides in Escherichia coli. We divided the project into
three modules: the DMAPP/IPP module, the steviol module, and the glycosyltransferase module. We synthesized the
genes for each module and assembled them using a method like NEB BioBrick assembly. These modules were then
inserted into vectors pACYCduet-1, PET21a, and PET28a. Under induction with IPTG, we expect to detect the
product of rebaudiosides.

![](https://static.igem.wiki/teams/5000/wiki/engineering/plus1.png)

## BUILD

1. The backbone of rebaudiosides is a terpenoid. Naturally, all terpenoids are built from C5 blocks:
   isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In eukaryotes, IPP and
   DMAPP are produced through the mevalonate (MVA) pathway while in prokaryotes they are produced through
   the methylerythritol-phosphate (MEP) pathway in. To increase the supply of isoprenoid we enhanced the
   expression of endogenous MEP pathway, three key enzymes for isoprenoid precursor,
   1-deoxyxylulose-5-phosphate synthase (DXS), farnesyl diphosphatesynthase (IspA) and isopentenyl
   diphosphate isomerase (IDI) were over overexpressed. (Kong et al., 2015)

   ![](https://static.igem.wiki/teams/5000/wiki/engineering/img01.png)
2. Since the terpenoids share the common C5 isoprenoid precursors, isopentenyl diphosphate (IPP) and its
   isomer dimethylallyl diphosphate (DMAPP), the commonly used methods include enhancing the endogenous MEP
   pathway or introducing exogenous MVA pathway were applied to enhance precursor supply. These natural
   pathways have many limitations, such as feedback inhibition of pathway intermediates or products and
   competing cofactor ATP, CTP, NADH, and NADPH, as well as precursors for IPP synthesis with central
   metabolism. To overcome these limitations, the isopentenol utilization pathway (IUP) were introduced. As
   shown the figure below, Isopentenol isomers were firstly phosphorylated to form isopentenyl
   monophosphate (IP) or dimethylallyl monophosphate (DMAP), and then they were convert to IPP or DMAPP by
   isopentenyl phosphate kinase (IPK). We incorporate the three genes, including SccK, AtiPK, idi into
   pACYCduet-1 for the biosynthesis of IPP and DMAPP from isopentenol. (Chatzivasileiou et al., 2018)

   ![](https://static.igem.wiki/teams/5000/wiki/engineering/img02.png)

   ![](https://static.igem.wiki/teams/5000/wiki/engineering/img03.png)
3. The steviol module consisting of six genes which are geranylgeranyl diphosphatase (GGPPS), ent-copayl
   diphosphate synthase (CDPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), NADPH-dependent
   cytochrome P450 reductase (CPR) and kaurenoic acid 13α-hydroxylase (KAH). We assemble them into Pet21a
   through NEB BioBrick assembly as shown below.(Wang et al., 2015)

   ![](https://static.igem.wiki/teams/5000/wiki/engineering/img04.png)
4. The steviol is further converted to different kinds of rebaudiosides through five glycosyl transferase
   (UGT82C2, UGT74G1, UGT76G1, UGT91D2w and EUGT11M(F379)). These genes were assembled into Pet28a and
   incorporated with the isoprenoid modules and steviol modules to achieve the biosynthesis of
   Rebaudiosides.(Wang et al., 2015, Lin et al., 2020)

   ![](https://static.igem.wiki/teams/5000/wiki/engineering/img05.png)

## TEST

1. To quantitatively analyze the production of steviol glycosides, we prepared a standard curve for
   Rebaudioside A, D and M. The analysis conditions are as follows: ThermoScientific Q Exactive Plus Mass
   Spectrometer; Column:  Agilent Zorbax C18, 5μm, 150 x 4.6 mm; Solvent:A, H2O+folic acid (0.1%); B, CH3CN
   + folic acid (0.1%), Gradient: 0 to 9 min, 20% to 50% solvent B; 9 to 10 min, 50% to 90% solvent B; 10
   to 11 min, 90% to 20% solvent B; 11 to 12 min, 20% B.

   ![](https://static.igem.wiki/teams/5000/wiki/engineering/plus2.png)
2. As a proof of concept, we construct three strains, all the strains derived from E.coli BL21(DE3)
   harboring different combination of plasmids. The plasmids are pACYCDuet-ispA-dxs-idi（MEP module） and
   pACYCDuet-Atipk-scck-idi（IUP module）， pET28a-UGT82C2-UGT74G1-UGT76G1-UGT91D2w-EUGT11M(F379)（glycosyl
   transferase module）, pET21a-GGPPS-KO-KS-CDPS-CPR-17αtr29CYP714A2（steviol module）. Strain A is a blank
   control, Strain B contains MEP module and steviol module, strain C contains IUP module and steviol
   module, strain D harbors the complete biosynthetic pathway for rebaudiosides which are IUP module,
   steviol module and glycosyl transferase module. After induction by 0.05 mM IPTG, these strain were
   cultivated in shake flask at 28℃ for 3 days. The culture were extracted by equivalent volume of
   n-butanol. After extraction, the organic phase was collected and dried under vacuum. Then they were
   dissolved by dimethyl sulfoxide and filtered. The samples were analyzed using ThermoScientific Q
   Exactive Plus Mass Spectrometer.

   ![](https://static.igem.wiki/teams/5000/wiki/engineering/img06.png)

## Learn

1. Through experimentation, we discovered that increasing the precursor process can effectively improve the
   yield of steviol glycosides. Introduction of IUP pathway is better than overexpression of the MEP
   pathway due to the influence of E. coli's inherent metabolic regulation. However, however both method
   can enhance the supply of precursor.
2. The activity of the glycosyltransferase needs improvement. Through experimentation, we observed the
   production of not only our intended product, Rebaudioside M, but also Rebaudioside A and Rebaudioside D
   as the final products.

## Reference

1. Kong, M., Kang, H. J., Kim, J. H., Oh, S. H., & Lee, P. C. (2015). Metabolic engineering of the Stevia
   rebaudiana
   ent-kaurene biosynthetic pathway in recombinant Escherichia coli. Journal of Biotechnology, 214, 95–102.
   https://doi.org/10.1016/j.jbiotec.2015.09.016
2. Chatzivasileiou, A. O., Ward, V. C., Edgar, S., & Stephanopoulos, G. (2018). Two-step pathway for
   isoprenoid
   synthesis. Proceedings of the National Academy of Sciences of the United States of America, 116(2),
   506–511.
   https://doi.org/10.1073/pnas.1812935116
3. Wang, J., Li, S., Xiong, Z., & Wang, Y. (2015). Pathway mining-based integration of critical enzyme parts
   for de
   novo biosynthesis of steviolglycosides sweetener in Escherichia coli. Cell Research, 26(2), 258–261.
   https://doi.org/10.1038/cr.2015.111
4. Lin, M., Wang, F., & Zhu, Y. (2020). Modeled structure-based computational redesign of a
   glycosyltransferase for
   the synthesis of rebaudioside D from rebaudioside A. Biochemical Engineering Journal, 159, 107626.
   https://doi.org/10.1016/j.bej.2020.107626

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

---

* [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)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

## Describe what your results mean

---

* 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

---

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

---

* [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)

{% endblock %}