iGEM 2021 — Team HZAU-China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HZAU-China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HZAU-China/Engineering

Loading

[Project](#item1)

[Description](https://2021.igem.org/Team%3AHZAU-China/Description)
[Design](https://2021.igem.org/Team%3AHZAU-China/Design)
[Implementation](https://2021.igem.org/Team%3AHZAU-China/Implementation)
[Proof of concept](https://2021.igem.org/Team%3AHZAU-China/Proof_Of_Concept)

[Wetlab](#item2)

[Safety](https://2021.igem.org/Team%3AHZAU-China/Safety)
[Protocol](https://2021.igem.org/Team%3AHZAU-China/Protocol)
[Part](https://2021.igem.org/Team%3AHZAU-China/Parts)
[Contribution](https://2021.igem.org/Team%3AHZAU-China/Contribution)
[Engineering](https://2021.igem.org/Team%3AHZAU-China/Engineering)
[Improvement](https://2021.igem.org/Team%3AHZAU-China/Improvement)
[Measurement](https://2021.igem.org/Team%3AHZAU-China/Measurement)

[Drylab](#item3)

[Model](https://2021.igem.org/Team%3AHZAU-China/Model)
[Hardware](https://2021.igem.org/Team%3AHZAU-China/Hardware)
[Software](https://2021.igem.org/Team%3AHZAU-China/Software)

[Human Practice](#item4)

[Human Practice](https://2021.igem.org/Team%3AHZAU-China/Human_Practices)
[Education](https://2021.igem.org/Team%3AHZAU-China/Education)
[Communication](https://2021.igem.org/Team%3AHZAU-China/Communication)
[Collaborations](https://2021.igem.org/Team%3AHZAU-China/Collaborations)
[Partnership](https://2021.igem.org/Team%3AHZAU-China/Partnership)

[About us](#item5)

[Team](https://2021.igem.org/Team%3AHZAU-China/Team)
[Attributions](https://2021.igem.org/Team%3AHZAU-China/Attributions)

![](https://static.igem.org/mediawiki/2021/2/29/T--HZAU-China--new-wet-pic.png)

![](https://static.igem.org/mediawiki/2021/8/80/T--HZAU-China--Engineering.png)

![](https://static.igem.org/mediawiki/2021/2/29/T--HZAU-China--new-wet-pic.png)

![](https://static.igem.org/mediawiki/2021/8/80/T--HZAU-China--Engineering.png)

# Engineering

This year, we have summited five new parts to the registry to prove our Engineering Success.

## ScGS

Gesomin synthase from *Streptomyces coelicolor* A3(2) (**ScGS**) is a single
726-amino acid protein which catalyzes the Mg2+ dependent conversion of farnesyl
diphosphate to a mixture including geosmin. ScGS is a bifunctional enzyme in which the
N-terminal domain catalyze the cyclization of FPP to form germacradienol, while the
C-terminal domain then convert this sesquiterpenoid product to **geosmin** [1].
As it shows in the following figure (**Figure 1**), the existence of ScGS in our chasis
was clearly proved by SDS-PAGE analysis.

![](https://static.igem.org/mediawiki/2021/0/02/T--HZAU-China--engineering-1.png
                                    )

**Figure 1.** SDS-PAGE analysis of ScGS with His-tag expression.

For further demonstration, we prepared the sample via headspace liguid-phase microextraction
(HS-LPME) and a gas chromatography-mass spectrometry (GC-MS) test was conducted. The results
given by GC-MS fairly shows the existence of geosmin in our culture (**Figure 2**), thus
prove the feasibility of the part.

![](https://static.igem.org/mediawiki/2021/0/02/T--HZAU-China--engineering-1.png
                                    )

**Figure 2.**Identification of geosmin by GC-MS. A. Total ion current
chromatogram of geosmin standard(Red Line) and extracted product(Blue line). B. Mass
spectrum of geosmin standard. C. Mass spectrum of the extracted product.

For more details, please click the link below: [http://parts.igem.org/Part:BBa\_K3733006](http://parts.igem.org/Part%3ABBa_K3733006)

## LTA

LTA is a novel antimicrobial peptide (AMP), whose design was based on combing the active
centers of a ride range of AMPs, including LL-37, YW12D, innate defense regulator 1, and
cathelicidin 2 with thymopentin or the active center of thymosin alpha 1 (Tα1). It could
neutralize Lipopolysaccharides (LPS), thus effectively blocking the downstream
inflammation
pathway.
As it shows in the following figure (**Figure 3**), the existence of LTA in our
chasis
was clearly proved by Tricine-SDS-PAGE analysis [2].

![](https://static.igem.org/mediawiki/2021/b/b6/T--HZAU-China--engineering-2.png
                                    )

**Figure 3.**Tricine-SDS-PAGE analysis of LTA.

We also confirmed the lytic activity of LTA by choosing *Salmonella Typhimurium*
SL1344
as our test strain. The result is shown below (**Figure 4**)

![](https://static.igem.org/mediawiki/2021/f/f2/T--HZAU-China--engineering-4.png
                                    )

**Figure 4.**OD600-Time curve of *Salmonella Typhimurium*
SL1344
in the presence or absence of LTA.

For more details, please click the link below: [http://parts.igem.org/Part:BBa\_K3733007](http://parts.igem.org/Part%3ABBa_K3733007)

## HyLα

HyLα is hybrid peptide of LL-37 and LTA. LL-37 and LTA are both affective antimicrobial
peptides (AMPs). successfully combine those two AMPs by connecting the C-terminate of LTA
with the N-terminate of LL-37 through GS-linker. It could neutralize Lipopolysaccharides
(LPS), thus effectively blocking the downstream inflammation pathway.
As it shows in the figure (**Figure 3**), the existence of HyLα in our chasis was clearly
proved by Tricine-SDS-PAGE analysis.
We also confirmed the lytic activity of HyLα by choosing *Salmonella Typhimurium*
SL1344 as our test strain. The result is shown below (**Figure 5**)

![](https://static.igem.org/mediawiki/2021/8/83/T--HZAU-China--engineering-5.png
                                            )

**Figure 5.**OD600-Time curve of *Salmonella Typhimurium* SL1344
in
the presence or absence of HyLα.

For more details, please click the link below: [http://parts.igem.org/Part:BBa\_K3733008](http://parts.igem.org/Part%3ABBa_K3733008)

## HepT

HepT is a toxin, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN)
superfamily, strongly inhibited cell growth in *S.oneidensis* and *Escherichia
coli*. The HepT toxin (HEPN-domain protein) could function as an RNase with a RX4-6H
active motif and cleave mRNA to inhibit cell growth [3].
The cytotoxicity of HepT is shown in the figure (**Figure 6**).

![](https://static.igem.org/mediawiki/2021/4/49/T--HZAU-China--engineering-6.png
                                            )

**Figure 6.** A. Turbidity change of the experimental group (induced by IPTG)
and
control group (without IPTG). B. The result of OD600 (-IPTG and +IPTG) presents the
cytotoxicity of HepT. The error bars are obtained from three independent
experiments.

For more details, please click the link below: [http://parts.igem.org/Part:BBa\_K3733010](http://parts.igem.org/Part%3ABBa_K3733010)

## RNA Thermometer\_NoHeat

This basic part is one of heat-repressible RNA thermosensor which could inhibit downstream
gene expression when the temperature is 37 ℃ but not affect downstream gene expression
significantly when the temperature is below 28 ℃.
The function of this RNA thermometer is based on the stem structure which was created by
taking the complement of the RNase-binding sequence. At low temperatures, the RNase-binding
sequence is protected by the stem structure and downstream gene could be expressed normally.
At high temperatures, the stem is destabilized, which allows RNase E (endogenic in *E.
coli*) to bind with the RNase-binding sequence and turn off downstream gene
expression [4].
We verified the function of this part in our project, the result is shown below (**Figure
7**).

![](https://static.igem.org/mediawiki/2021/4/4b/T--HZAU-China--engineering-7.png
                                            )

**Figure 7.** A. The comparison photo of the experimental group (toxin system)
and
control group incubated at both 37 ℃ and 28 ℃ for 12 hours. NoHeat means the
experimental group (toxin system). Control means the control group. B. The specific
OD600 data of the experimental group and control group.

For more details, please click the link below: [http://parts.igem.org/Part:BBa\_K3733011](http://parts.igem.org/Part%3ABBa_K3733011)

#### Reference:

[1] Harris G G, Lombardi P M, Pemberton T A, et al. Structural Studies of Geosmin
Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That
Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence[J]. Biochemistry, 2015,
54(48): 7142-7155.

[2]Zhang L, Wei X, Zhang R, et al. Design and development of a novel peptide for

treating intestinal inflammation [J]. Frontiers in immunology, 2019, 10: 1841.

[3] Yao J, Zhen X, Tang K, et al. Novel polyadenylylation-dependent neutralization
mechanism of the HEPN/MNT toxin/antitoxin system[J]. Nucleic acids research, 2020,
48(19): 11054-11067.

[4] Hoynes-O'Connor A, Hinman K, Kirchner L, et al. De novo design of heat-repressible
RNA thermosensors in E. coli[J]. Nucleic acids research, 2015, 43(12): 6166-6179.

* [Engineering](#Engineering)
  + [ScGS](#ScGS)
  + [LTA](#LTA)
  + [HyLα](#HyLα)
  + [RNA Thermometer\_NoHeat](#RNA Thermometer_NoHeat)

![](https://static.igem.org/mediawiki/2021/0/06/T--HZAU-China--HZAU.png)

![](https://static.igem.org/mediawiki/2021/7/75/T--HZAU-China--snapgene%281%29.png)

![](https://static.igem.org/mediawiki/2021/2/2a/T--HZAU-China--GeneScript.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HZAU-China/Experiments

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HZAU-China/Results

# Results

You can describe the results of your project and your future plans here.

### 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.

### Inspiration

See how other teams presented their results.

* [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)