iGEM 2022 — Team bnsc-china

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{% 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 %}
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## What should this page contain?

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* 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

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* [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)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Our Engineering Success on Cross-species Promoters Design{% endblock %}
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In our design, there is a module on cross-species gene expression in which we want to design synthetic promoters that work in both prokaryotes as well as yeast.

Currently, the synthetic promoters available in the literature are limited and have a small dynamic range, limiting the testing of cross-species gene expression. To address this issue, we chose to cross the J23 family of constitutive promoters (J23119, J23100, J23104, J23113) as well as plac in prokaryotes with several artificially designed promoters (TE1, TE2, TE3, TE4, TE5) in yeast recently reported in the literature. The bacterial constitutive promoter is loaded upstream of the yeast promoter to form a hybrid promoter.

We tested these combinations in brewer's yeast as well as bacteria, using the TEF1 promoter, which is capable of expression in both prokaryotic and eukaryotic forms, as a control. The results showed that both in yeast and in bacteria, these promoters stably drove gene expression and showed a decreasing trend consistent with data reported in the literature. This set of results suggests that our engineering of this module was successful.

![We hope to develop new synthetic promoters for species expression. A: plasmids used to test species compatibility, with synthetic promoters in gray boxes, where the bacterial promoter of the J23 family is located upstream of the yeast promoter. B: synthetic promoters working in yeast, with TEF1 as a control. C: synthetic promoters working in E. coli, with TEF1 as a control.](https://static.igem.wiki/teams/4237/wiki/engineering/engineering-promoter.png "Schema and results of cross-species promoters")

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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 %}Boldly assume, carefully verify.{% endblock %}
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## Golden gate collection that enables rapid production validation across strains

The Marburg Collection, proposed by the 2018 Marburg team, has well-established prokaryotic expression elements. To
expand its cross-species functionality, we propose ONCE\_Collection with the following modifications:

* 1. introduce hybrid promoters compatible with prokaryotes and brewer's yeast to the Promoter module;
* 2. add short synthetic terminators for yeast to the terminator module;
* 3. add elements for splice transfer and replication in yeast to the ORI module;
* 4. add enzymes for rapid genome integration to the CDS module;
* 5. add recombinase recognition module and yeast homologous recombination module to the connector module;
* 6. add nutritional defect screening tools to the antibiotic resistance module;
* 7. introduce inducible promoters and corresponding regulators.

### Promoters and terminaors that are compatible with yeast and prokaryotic organisms

We crossed the constitutive promoters of the J23 family (J23119, J23100, J23104, J23113) in prokaryotes as well as
plac with several artificially designed promoters (TE1, TE2, TE3, TE4, TE5) in yeast that have been recently
reported in the literature. The bacterial constitutive promoter is loaded upstream of the yeast promoter to form a
hybrid promoter. We tested these combinations in brewer's yeast as well as bacteria and found that these promoters
stably drove gene expression in both yeast and bacteria, and showed a decreasing trend consistent with data reported
in the literature. These data suggest that our design worked, and although these synthetic promoters may differ in
intensity from when they work alone, at the functional level this can be left out of the discussion.

![Figure 1 Plasmid profiles and results for testing synthetic promoters in E. coli and Saccharomyces cerevisiae.](https://static.igem.wiki/teams/4237/wiki/results/fig1.png)

### ORI compatible with splice transfer, yeast shuttle

The combination of OriT with other ORIs allows ORIs of other copy numbers to shuttle between E. coli and non-model
bacteria. Further addition of CEN/PK or 2u ORIs capable of replication in yeast to the backbone would enable
shuttling in yeast.

![Figure 2 ORI combinations that can be splice transferred and work in yeast and E. coli.](https://static.igem.wiki/teams/4237/wiki/results/fig2.png)

We first constructed the three combinations as above and tested them with in DH5a. It can be found that for each
ORI, together with OriT as well as CEN/PK replicas, clones can be successfully constructed and none of them affect
the expression function in E. coli.

![Figure 3 Characterization data of different ORI combinations in E. coli DH5α.](https://static.igem.wiki/teams/4237/wiki/results/fig3.png)

All OriT combinations were successfully transferred into Nissle 1917. The combination of these
five ORIs, together with pBBR1, enables a broader spectrum of non-model organism gene expression.

![Figure 4 Splice transfer ORI working in Nissle 1917](https://static.igem.wiki/teams/4237/wiki/results/fig4.png)

### Efficient strategies for rapid genome integration

We tested PhiC31 with different expression intensities and the same incubation time, the TnsABCE and Cre at
different expression intensities and the same incubation time.

![Fig 5 Integration efficiency of different recombinant enzymes at different expression intensities](https://static.igem.wiki/teams/4237/wiki/results/fig5.png)

### inducible promoter and the corresponding regulator

As we mentioned in the Human practice module, we systematically characterized the leaked expression of the
marionette promoter for better metabolic engineering applications in *V.nat*. First, we mounted the
device of the inducible promoter on a plasmid with ORI pBBR1 and transferred it into *V.nat*. We
found that the leakage expression of PbetI was moderate, and we judged that it was more suitable for Plac for the
leakage expression of metabolic pathways in *V.nat*.

![Fig 6 Leaky of each inducible promoter in Vibiro natriegens](https://static.igem.wiki/teams/4237/wiki/results/fig6.png)

## A general strategy for the construction of high FPP production and downstream terpenoid chassis strains

We plan to improve FPP precursor synthesis as well as downstream product production by exogenously expressing the
MVA pathway, The lycopene production was increased approximately 13-fold compared to our most primitive
strain.

![Fig 7 Validation of the strategy for high yield of FPP and downstream terpenoids](https://static.igem.wiki/teams/4237/wiki/results/fig8.png)

The strategy we designed has great potential for efficient synthesis of terpenoids.

### FPP sensing system based on split fluorescent protein

Although we used lycopene synthesis to characterize FPP supply, given the complex FPP metabolic network within
bacteria, we further developed a sensing system that directly binds FPP and emits fluorescence (FPR) and validated
the feasibility of this system in E. coli DH5α as well as the optimal detection time.

Limited by the lack of available FPP monomers, we used DH5α that had been characterized with the MVA pathway
transferred as a positive control and DH5α with only the MVA pathway as a negative control. As shown in the result
graph, FPPR has only a small background fluorescence intensity in wild-type DH5α, which is due to the fact that E.
coli naturally contains the MEP pathway for FPP synthesis and low levels of FPP concentration exist in the bacteria.
In contrast, the addition of the MVA pathway greatly increased the fluorescence signal intensity of FPPR
(~3.6-fold), demonstrating the responsiveness of our reporter system to the concentration of FPP.

![Figure 8 Validation of strategy for high yield FPP and downstream terpenoids](https://static.igem.wiki/teams/4237/wiki/results/fig9.png)

### Production of (-)-α-bisabolol

In order to produce (-)-α-bisabolol more efficiently, we verified the expression of bacterial ccBOS using Coomassie Blue staining and found that although ccBOS expression was higher at 30°C compared to bacteria grown at 25°C, it was mostly concentrated in the cellular sediment, which we hypothesized was due to too rapid synthesis of ccBOS and failure to fold correctly. We therefore lowered the fermentation temperature to 25°C, and this time we achieved a 5.7-fold increase in yield, demonstrating that our optimized strain as well as fermentation conditions can be used for efficient (-)-α-bisabolol production.

![Figure 11 Coomassie Blue staining of ccBOS](https://static.igem.wiki/teams/4237/wiki/results/ccbos-cbs.png)
![Figure 12 Yield of (-)-α-bisabolol under different fermentation conditions](https://static.igem.wiki/teams/4237/wiki/results/ecoli-abis.png)

## Sea water cultivation of *V.nat*

We use non-sterilized fermentation of *Vibrio natriegens* with seawater, to systematically verify the
feasibility of the project, we plan to collect seawater from China Major sea areas and two major marine economic
species farming wastewater。for non-nterilized fermentation.

This experiment has been submitted to check-in form and approved.We ensure that the obtained seawater does not
violate local laws and regulations, the water source does not involve the epidemic area. The water intake of each
water source shall not exceed four liters and the intaking will not affect the local natural ecology. We guarantee
that the obtained seawater is safely and properly stored and used in the laboratory and treated in a biosafety
manner after the experiment.

| Sampling name | Sampling locations | longitude and latitude |
| --- | --- | --- |
| Hainan | Baishamen Park, Meilan District, Haikou City | 110.341546,20.076778 |
| Xiamen | White City Beach, Siming District, Xiamen | 118.109729,24.438016 |
| Shenzhen | Shenzhen Bay, Nanshan District, Shenzhen | 114.003481,22.52837 |
| Yancheng | Moon Bay, Binhai County, Yancheng City | 120.274268,34.316129 |
| H.americanus; P.trituberculatus aquaculture wastewater | Yangpu District, Shanghai | 121.479965,31.238646 |
| Dalian | Dayaowan Port, Jinzhou District, Dalian | 121.85914,39.029128 |

![Figure12 Sampling site location](https://static.igem.wiki/teams/4237/wiki/results/fig11.png)

To systematically validate the potential of non-sterile fermentation of sodium-demanding Vibrio seawater, we used
seven natural seawater or marine aquaculture wastewater as substrates, adding carbon sources under normal culture
conditions without additional sodium chloride. Depending on whether the formulated medium was added with or without
kana, the autoclaved steam was non-sterile or non-sterile divided into four groups. Bacterial growth is monitored in
real time by testing OD600.

![Fig13A General overview of seawater fermentation growth curves](https://static.igem.wiki/teams/4237/wiki/results/fig12a.png)
![Figure13B Culture curve of Shenzhen seawater](https://static.igem.wiki/teams/4237/wiki/results/fig12b.png)
![Figure13C Culture curve of Hainan seawater](https://static.igem.wiki/teams/4237/wiki/results/fig12c.png)
![Figure13D Culture curve of P.trituberculatus farming wastewater](https://static.igem.wiki/teams/4237/wiki/results/fig12d.png)
![Figure13E: Culture curve of H.americans farming wastewater](https://static.igem.wiki/teams/4237/wiki/results/fig12e.png)
![Figure13F: Culture curve of Shenzhen seawater](https://static.igem.wiki/teams/4237/wiki/results/fig12f.png)
![Figure13G: Culture curve of Xiamen seawater](https://static.igem.wiki/teams/4237/wiki/results/fig12g.png)
![Figure13H: Culture curve of Yancheng seawater](https://static.igem.wiki/teams/4237/wiki/results/fig12h.png)
![Figure13I: Culture curve of Selected seawater compared with LBV2](https://static.igem.wiki/teams/4237/wiki/results/fig12i.png)

The growth curves of water samples from different regions were significantly different, and we speculate that this
may be due to differences in the physicochemical properties of seawater in different regions. To further investigate
the reason, we examined the salinity, pH, nitrite, phosphate, Ca2+, Mg2+, total hardness, total alkalinity of all
water samples, and the results are summarized below, which in turn facilitate subsequent reference for the selection
of seawater non-sterilized fermentation water sources.

![](https://static.igem.wiki/teams/4237/wiki/results/fig13a.png)
![ Figure14 The physicochemical properties (salinity, pH, nitrite, phosphate, Ca2+, Mg2+, total hardness, total alkalinity) of different seawater](https://static.igem.wiki/teams/4237/wiki/results/fig13b.png)

## References

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8. Greco F V, Pandi A, Erb T J, et al. Harnessing the central dogma for stringent multi-level control of gene expression[J]. Nature communications, 2021, 12(1): 1-11.
9. Hoff J, Daniel B, Stukenberg D, et al. Vibrio natriegens: an ultrafast‐growing marine bacterium as emerging synthetic biology chassis[J]. Environmental microbiology, 2020, 22(10): 4394-4408.
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11. Glasgow, Anum A., Yao-Ming H, Daniel J. M, et al. Computational Design of a Modular Protein Sense-Response System[J]. Science 2019, 366 (6468): 1024–28.

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