iGEM 2020 — Team QHFZ-China

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:QHFZ-China/Notebook

* [![Back to main page](https://static.igem.org/mediawiki/2020/3/36/T--QHFZ-China--%E9%98%9F%E5%BE%BD.jpg "Back to main page")](https://2020.igem.org/Team%3AQHFZ-China)
* [Project](https://2020.igem.org/Team%3AQHFZ-China/Description)
* [Model](https://2020.igem.org/Team%3AQHFZ-China/Model)
* [Human Practices](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
* [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
* [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* [Judging](https://igem.org/2020_Judging_Form?id=3457)
* [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)

* + [Description](https://2020.igem.org/Team%3AQHFZ-China/Description)
  + [Design](https://2020.igem.org/Team%3AQHFZ-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AQHFZ-China/Proof_Of_Concept)
  + [Results](https://2020.igem.org/Team%3AQHFZ-China/Results)
  + [Implementation](https://2020.igem.org/Team%3AQHFZ-China/Implementation)
  + [Experiments](https://2020.igem.org/Team%3AQHFZ-China/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AQHFZ-China/Notebook)
* + [Modeling](https://2020.igem.org/Team%3AQHFZ-China/Model)
  + [Protein](https://2020.igem.org/Team%3AQHFZ-China/Protein)
* + [Intergrated](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQHFZ-China/Education)
* + [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
  + [Contribution](https://2020.igem.org/Team%3AQHFZ-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AQHFZ-China/Engineering)
  + [Collection](https://2020.igem.org/Team%3AQHFZ-China/Part_Collection)
* + [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* + [Judging](https://igem.org/2020_Judging_Form?id=3457)
* + [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AQHFZ-China/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3AQHFZ-China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQHFZ-China/Partnership)
  + [Acknowledgement](https://2020.igem.org/Team%3AQHFZ-China/Acknowledgement)

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副页面

# Notebook

Part 1: Experimental Records
    Part 2: Notable Advances

## Notable Advances

### On July 2nd, we finished constructing pet28a-modification. On July 21st,we did our lyophilization experiment for the first time. On July 29th, we finished constructing pet28a-sfGFP. On August 10th, we finished constructing pet28a-modification-106094 and pet28a-modification-107838. On August 18th, we got the first positive result for our lyophilization experiment. On August 24th, we finished constructing pet28a-modification-sfGFP and pet28a-modification-33020-106094. On August 27th, we finished constructing pet28a-modification-J100/J107/J109-sfGFP. On September 17th,we confirmed that the survival rate could improve with the increase of 106094’s expression. On September 23rd,we confirmed that pet28a-modification-33020-106094 could improve the resistance of bacteria under lyophilization environment. On September 25th,we finished constructing pet28a-modification-J100-106094-pdtA.

## Experimental records

[The record of molecular cloning](https://static.igem.org/mediawiki/2020/1/1b/T--QHFZ-China--Notebook1.pdf)

[The record of freeze-drying, fluorescence detection and SDS-PAGE](https://static.igem.org/mediawiki/2020/9/91/T--QHFZ-China--Notebook2.pdf)

[The record of cytobiology](https://static.igem.org/mediawiki/2020/f/f0/T--QHFZ-China--Notebook3.pdf)

# SPONSORS

WE APPRECIATE ALL OF YOU.

[![mathworks](https://static.igem.org/mediawiki/2020/1/1f/T--QHFZ-China--mathworks.png)](https://www.mathworks.com)
[![geneious](https://static.igem.org/mediawiki/2020/6/67/T--QHFZ-China--geneious.png)](https://www.geneious.com)
[![snapgene](https://static.igem.org/mediawiki/2020/4/43/T--QHFZ-China--snapgene.png)](https://www.snapgene.com)
[![tsinghua](https://static.igem.org/mediawiki/2020/1/10/T--QHFZ-China--tsinghua.jpg)](http://www.qhfz.edu.cn)
[![zeno](https://static.igem.org/mediawiki/2020/9/9e/T--QHFZ-China--zeno.jpg)](https://mp.weixin.qq.com/s?__biz=MzI2MDYzNzE3MA==&mid=100000382&idx=1&sn=846a73f9464294d9a13eb88becfb2e71&chksm=6a67eae35d1063f590a71cdc82234a11ff77c116f28ec2a809e63c82b03d16bac9fe41924acd&mpshare=1&scene=1&srcid=1028Y6lu2yBbhHFbIsfaLmon&sharer_sharetime=1603842347682&sharer_shareid=9ab52608d26284723c47930bc32142bb&key=fe9a3435302fce659fb6e57197f58051b8bed0826563a142eb049467fc7e98cf542d49ce9abdb10a37a86676a16a25346545c09e5b8b5343c15bd31df2bb914aa4a491717cf461be0c8810a4e2ecec7eb3d93a73bd7f28e5a82584278d20fdd98de9f3c7b0560d7e5d3c2c2671d0e6a3d19781febe2c5224da715e7fc5f4af66&ascene=1&uin=MTExNzMyMDQ5OQ%3D%3D&devicetype=Windows+10+x64&version=6300002f&lang=zh_CN&exportkey=A6zy9oj4tjeXUUlCzYB7R7k%3D&pass_ticket=g4e9OqLU5NiTOjz%2FzgDW45YCOA%2FYTo01G%2B2WhcqPHBnyNRgZRQMDwCqZvprEaLac&wx_header=0)
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# Team:QHFZ-China/Engineering

* [![Back to main page](https://static.igem.org/mediawiki/2020/3/36/T--QHFZ-China--%E9%98%9F%E5%BE%BD.jpg "Back to main page")](https://2020.igem.org/Team%3AQHFZ-China)
* [Project](https://2020.igem.org/Team%3AQHFZ-China/Description)
* [Model](https://2020.igem.org/Team%3AQHFZ-China/Model)
* [Human Practices](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
* [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
* [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* [Judging](https://igem.org/2020_Judging_Form?id=3457)
* [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)

* + [Description](https://2020.igem.org/Team%3AQHFZ-China/Description)
  + [Design](https://2020.igem.org/Team%3AQHFZ-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AQHFZ-China/Proof_Of_Concept)
  + [Results](https://2020.igem.org/Team%3AQHFZ-China/Results)
  + [Implementation](https://2020.igem.org/Team%3AQHFZ-China/Implementation)
  + [Experiments](https://2020.igem.org/Team%3AQHFZ-China/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AQHFZ-China/Notebook)
* + [Modeling](https://2020.igem.org/Team%3AQHFZ-China/Model)
  + [Protein](https://2020.igem.org/Team%3AQHFZ-China/Protein)
* + [Intergrated](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQHFZ-China/Education)
* + [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
  + [Contribution](https://2020.igem.org/Team%3AQHFZ-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AQHFZ-China/Engineering)
  + [Collection](https://2020.igem.org/Team%3AQHFZ-China/Part_Collection)
* + [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* + [Judging](https://igem.org/2020_Judging_Form?id=3457)
* + [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AQHFZ-China/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3AQHFZ-China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQHFZ-China/Partnership)
  + [Acknowledgement](https://2020.igem.org/Team%3AQHFZ-China/Acknowledgement)

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# Engineering Success

We are lucky to have the chance to get into lab this year, so we proved our concept, as well as confirming
the engineering success by our project design and experiment data. The content are shown on the [Description](https://2020.igem.org/Team%3AQHFZ-China/Description) and [Proof Of Concept](https://2020.igem.org/Team%3AQHFZ-China/Proof_Of_Concept)
pages.
As a result, to achieve this criterion, we follow the judging guidance and describe here a new part
which is constructed and measured by us this year.

## New part: [BBa\_K3457009](http://parts.igem.org/Part%3ABBa_K3457009) CAHS 89226 with a 6X His tag

We have uploaded it onto the on the Registry ([http://parts.igem.org/Part:BBa\_K3457009](http://parts.igem.org/Part%3ABBa_K3457009)), now we will carry the content here for
convenient check.

    This biological is the CDS sequence of Cytosolic-abundant heat soluble protein 89226,
also called CAHS 89226. It is a kind of Tardigrade intrinsically Disordered Protein (TDP). It is a heat
soluble protein found from Tardigrade *Hypsibius dujardini* in 2017 [1]. Tardigrade, also
called water bear, is a kind of tenacious organism. It can survive extreme environment, such as desiccation,
freeze and vacuum. The super capacity of Tardigrade partially owes to TDPs. CAHS 89226 has been proven to
protect the activity of purified proteins [1]. Here we found that expressing this protein can help bacteria survive the freeze-drying process, and then the resultant dry bacteria powder can be stored for a long time at room temperature.

### Design

![](https://static.igem.org/mediawiki/2020/9/90/T--QHFZ-China--ES_1.png)

Figure 1. The Schematic cartoon of the DNA construct of BBa\_K3457009.

    This part is the CDS of CAHS 89226. We obtained the sequence from the research paper in 2017 [1]. With the help of a company, we optimized the sequence so that the part is suitable to work in E. coli, and there is no biobrick restriction enzyme cutting site in it. Moreover, we added a 6x His tag at the N-terminal of CAHS 89226, so it can be easily detected by Western Blot and be purified by Ni-chelating affinity chromatography (Fig.1). This year, the part is used in the composite part  [BBa\_K3457032](http://parts.igem.org/Part%3ABBa_K3457032).

### Documentation:

#### Introduction:

    This year, we tried to introduce a new bio preservation method. We used freeze-drying to make the engineered into dry powder. Then the powder can be stored at room temperature for a long time. This method can store bacteria get rid of ultra-low temperature freezer to promote the practical application of engineered bacteria out of the laboratory. However, the stresses during freeze-drying and subsequent dry storage, including freeze, dry, and vacuum, are lethal to bacteria. We use TDPs, including CAHS 89226, to help bacteria survive the situation.

#### Protocol:

    To test the effect of CAHS 89226, we modified a frequently and widely used vector,
pet28a+ and put this part into it (Fig. 2). Then we transformed the plasmid into *E. coli* BL21 strain.

![](https://static.igem.org/mediawiki/2020/8/85/T--QHFZ-China--ES_2.png)

Figure 2. The Schematic cartoon of the vector.

    Then we used the following protocols to verify its function (Fig. 3):

![](https://static.igem.org/mediawiki/2020/a/a6/T--QHFZ-China--proof_1.jpg)

Figure 3. Experiment protocol.

【Day 1】Induction culture
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake
them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in
step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃
overnight.
【Day 2】Freeze-dried
(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the
experiment.
(2) Use spectrophotometer to measure the OD600 of the bacteria solution, OD600 = 1
equals to 109 cells. If the OD600 value is between 0.1 and 1, There is a linear
relationship between OD600 and bacterial density. Calculate the volume of bacterial solution for
109 cells by using the formula V = 100 / (OD600 × Dilution ratio).
(3) Take out a measured amount of 109 cells and centrifuge it at 8000 rpm for 3 min. Then pour
out the supernatant.
(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.
(5) Take off the cover of the tube and put the bacteria into the cold trap. Open the compressor of the
lyophilization machine and freeze the shake tube for 2 h at -70℃.
(6) Put the caky bacteria solution into the drying chamber of the lyophilization machine. Open the vacuum
pump to dry it in vacuum for 6h at 1 Pa vacuum degree.
(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room
temperature.
【Day 3】Room temperature storage
【Day 4】Detect the survival rate
(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.
(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria
solution on the LB plate.
(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after
several gradient dilutions.
(4) Culture the bacteria overnight at 37℃.
【Day 5】Cell Count
(1) Take out the LB plate and take photos to record experimental results.
(2) Use the automatic cell counting function of Image J to count the colone number on the LB plate, then
compare the results between each group.

#### Results:

    First, through a reporter, we confirmed that the plasmid sfGFP can normally express exogenous proteins in the *E. coli* BL21 strain (Fig. 4). Via SDS-PAGE,we observed the band of CAHS89226 and further verified the successful expression.
further verified the successfully expression (Fig.5).

![](https://static.igem.org/mediawiki/2020/9/9c/T--QHFZ-China--ES_4.png)

Figure 4. Via a reporter, sfGFP, the expression function of the modified pet28a vector was verified.

![](https://static.igem.org/mediawiki/2020/5/5b/T--QHFZ-China--ES_5.png)

Figure 5. CAHS 89226 was expressed as expected.

    Then, by freeze-drying and recover experiment, we found that compared with the
control group (sfGFP), the bacteria expressing CAHS 89226 had a better survival rate (Fig. 6). We placed an
enlarged picture as well (Fig. 7).

![](https://static.igem.org/mediawiki/2020/9/9b/T--QHFZ-China--ES_6.png)

Figure 6. CAHS 89226 protected bacteria during freeze-drying and subsequent dry storage.

![](https://static.igem.org/mediawiki/2020/0/03/T--QHFZ-China--ES_7.jpg)

Figure 7. An enlarged picture of fig. 6.

#### Summary:

    CAHS 89226 helps the bacteria survive freeze-drying and subsequent dry storage. To
achieve that, you need only to transform the sequence into your bacteria. You can also use the part to
express and purify CAHS 89226 through Ni-chelating affinity chromatography. The purified CAHS 89226 can be
used to protect protein products.

#### Reference:

[1] Boothby, T.C., Tapia, H., Brozena, A.H., Piszkiewicz, S., Smith, A.E., Giovannini, I., Rebecchi, L.,
Pielak, G.J., Koshland, D., and Goldstein, B. (2017). Tardigrades Use Intrinsically Disordered Proteins to
Survive Desiccation. Mol Cell 65, 975-984 e975.

## Note

Though only one new part is enough to achieve the criterion, we constructed more than one this year. For example, [BBa\_K3457059](http://parts.igem.org/Part%3ABBa_K3457059) Dsup for human cells. We have uploaded it onto the on the Registry and put the list on our [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts) page.

# SPONSORS

WE APPRECIATE ALL OF YOU.

[![mathworks](https://static.igem.org/mediawiki/2020/1/1f/T--QHFZ-China--mathworks.png)](https://www.mathworks.com)
[![geneious](https://static.igem.org/mediawiki/2020/6/67/T--QHFZ-China--geneious.png)](https://www.geneious.com)
[![snapgene](https://static.igem.org/mediawiki/2020/4/43/T--QHFZ-China--snapgene.png)](https://www.snapgene.com)
[![tsinghua](https://static.igem.org/mediawiki/2020/1/10/T--QHFZ-China--tsinghua.jpg)](http://www.qhfz.edu.cn)
[![zeno](https://static.igem.org/mediawiki/2020/9/9e/T--QHFZ-China--zeno.jpg)](https://mp.weixin.qq.com/s?__biz=MzI2MDYzNzE3MA==&mid=100000382&idx=1&sn=846a73f9464294d9a13eb88becfb2e71&chksm=6a67eae35d1063f590a71cdc82234a11ff77c116f28ec2a809e63c82b03d16bac9fe41924acd&mpshare=1&scene=1&srcid=1028Y6lu2yBbhHFbIsfaLmon&sharer_sharetime=1603842347682&sharer_shareid=9ab52608d26284723c47930bc32142bb&key=fe9a3435302fce659fb6e57197f58051b8bed0826563a142eb049467fc7e98cf542d49ce9abdb10a37a86676a16a25346545c09e5b8b5343c15bd31df2bb914aa4a491717cf461be0c8810a4e2ecec7eb3d93a73bd7f28e5a82584278d20fdd98de9f3c7b0560d7e5d3c2c2671d0e6a3d19781febe2c5224da715e7fc5f4af66&ascene=1&uin=MTExNzMyMDQ5OQ%3D%3D&devicetype=Windows+10+x64&version=6300002f&lang=zh_CN&exportkey=A6zy9oj4tjeXUUlCzYB7R7k%3D&pass_ticket=g4e9OqLU5NiTOjz%2FzgDW45YCOA%2FYTo01G%2B2WhcqPHBnyNRgZRQMDwCqZvprEaLac&wx_header=0)
[![tide](https://static.igem.org/mediawiki/2020/6/62/T--QHFZ-China--tide.jpg)](http://www.tidepharm.com)
[![youcare](https://static.igem.org/mediawiki/2020/5/5a/T--QHFZ-China--youcare.jpg)](http://www.youcareyk.com)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:QHFZ-China/Experiments

* [![Back to main page](https://static.igem.org/mediawiki/2020/3/36/T--QHFZ-China--%E9%98%9F%E5%BE%BD.jpg "Back to main page")](https://2020.igem.org/Team%3AQHFZ-China)
* [Project](https://2020.igem.org/Team%3AQHFZ-China/Description)
* [Model](https://2020.igem.org/Team%3AQHFZ-China/Model)
* [Human Practices](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
* [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
* [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* [Judging](https://igem.org/2020_Judging_Form?id=3457)
* [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)

* + [Description](https://2020.igem.org/Team%3AQHFZ-China/Description)
  + [Design](https://2020.igem.org/Team%3AQHFZ-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AQHFZ-China/Proof_Of_Concept)
  + [Results](https://2020.igem.org/Team%3AQHFZ-China/Results)
  + [Implementation](https://2020.igem.org/Team%3AQHFZ-China/Implementation)
  + [Experiments](https://2020.igem.org/Team%3AQHFZ-China/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AQHFZ-China/Notebook)
* + [Modeling](https://2020.igem.org/Team%3AQHFZ-China/Model)
  + [Protein](https://2020.igem.org/Team%3AQHFZ-China/Protein)
* + [Intergrated](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQHFZ-China/Education)
* + [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
  + [Contribution](https://2020.igem.org/Team%3AQHFZ-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AQHFZ-China/Engineering)
  + [Collection](https://2020.igem.org/Team%3AQHFZ-China/Part_Collection)
* + [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* + [Judging](https://igem.org/2020_Judging_Form?id=3457)
* + [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AQHFZ-China/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3AQHFZ-China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQHFZ-China/Partnership)
  + [Acknowledgement](https://2020.igem.org/Team%3AQHFZ-China/Acknowledgement)

[TOP](#top)

副页面

# Experiments

#nbsp;#nbsp;#nbsp;#nbsp;

Though the COVID-19 affect our experimental process, we still feel fortunate to have the change doing our experiment instead of just gathering online. In this page, we'll show the protocol of the experiments we do this year and also the process on how we do the lyophilization.

## Molecular Cloning

【1】PCR
Primers were designed by snapgene software and synthesized at GENEWIZ Company. PCR was performed by following the manuals of PrimeSTAR Max Premix (2X) (Taraka, R045) or 2 X EasyTaq PCR SuperMix (+ dye) (Transgene, M256). The former was used for construction and the latter was used for identification.

【2】Gel extraction
DNA fragment were separated by agarose gel electrophoresis and then recycled by TIANgeo Midi Purification Kit (TIANGEN, DP209-02) or AxyPrep DNA gel Extraction Kit (AXYGEN, AP-GX-250).

【3】Gibson
Gibson was performed by following the manual of pEASY- Basic Seamless Cloning and Assembly Kit (Transgene, CU201).

【4】Endonuclease digestion and ligation
The endonucleases and T4 ligase were bought from NEB Company and the experiments were performed by following instructions.

【5】Transformation
Transformation was performed by following the manuals of Trans5α Chemically Competent Cell (Transgene, CD201) or Trans1-T1 Phage Resistant Chemically Competent Cell (Transgene, CD501).

【6】Plasmid extraction
Bacteria were cultured in lysogeny broth (LB) at 37℃ overnight and plasmid extraction was performed by following the manuals of TIANprep Mini Plasmid Kit (TIANGEN, DP103-02) or AxyPrep Plasmid Miniprep Kit (AXYGEN, AP-MN-P-250).

【7】Sequencing
Sanger sequencing was done by GENEWIZ Company and snapgene software is used to align the sequences.

## Lyophilization Process

【Day 1】Induction culture
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add 2 mM iPTG into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
【Day 2】Freeze-dried
(1) If fluorescence induced by the iPTG is detectable in the control group (GFP), continue conducting the experiment.
(2) Use spectrophotometer to measure the OD600 of the bacteria solution, OD600 = 1 equals to 109 cells. If the OD600 value is between 0.1 and 1, There is a linear relationship between OD600 and bacterial density. Calculate the volume of bacterial solution for 109 cells by using the formula V = 100 / (OD600 × Dilution ratio).
(3) Take out a measured amount of 109 cells and centrifuge it at 8000 rpm for 3 min. Then pour out the supernatant.
(4) Resuspend the bacteria in a 15 mL tube with pre-refrigerated 100 μL 3% glucose solution.
(5) Take off the cover of the tube and put the bacteria into the cold trap. Open the compressor of the lyophilization machine and freeze the shake tube for 2 h at -70℃.
(6) Put the caky bacteria solution into the drying chamber of the lyophilization machine. Open the vacuum pump to dry it in vacuum for 6h at 1 Pa vacuum degree.
(7) Turn off the vacuum pump, place it at seal box filled with silica-gel desiccant a for 2 days at room temperature.
【Day 3】Room temperature storage
【Day 4】Detect the survival rate
(1) Add 1 mL of sterile water to the tube, vortex for 15 s, placed it at room temperature for 10 min.
(2) Adjust the density of the bacteria solution by gradient dilution, then spread 100 μL of the bacteria solution on the LB plate.
(3) If the density above is not suitable, take 100μL of the solution and spread it on the LB plate after several gradient dilutions.
(4) Culture the bacteria overnight at 37℃.
【Day 5】Cell Count
(1) Take out the LB plate and take photos to record experimental results.
(2) Use the automatic cell counting function of Image J to count the colone number on the LB plate, then compare the results between each group.

![](https://static.igem.org/mediawiki/2020/c/cd/T--QHFZ-China--exp-1.png)

## Protein

SDS-PAGE
Bacteria are cultured overnight. OD600 is tested. The same nubmer
(3~5 x 10e9) bacteria of different group is collected. Then the cells are lysed by 100~200 μL 4% SDS for 5 min in room temperature, then 10 min at 95℃. Then 6x loading is added. The SDS-PAGE was carried with a 12% precast polyacrylamide gel. Coomassie bright blue stain is used to observa the bands.

## Cytology experiments

【1】Cell culture
Human HeLa cells were cultured in complete DMEM (CD) medium (89% High-glucose DMEM, 10% fetal bovine serum (FBS) and 1% Penicillin / streptomycin). Generally, the medium was changed every day. Tyrisin was used for passage. Usually, 1 × 105 cells were seeded in every well of 6-well plate.

【2】Transient transfection
Plasmid was extracted by TIANprep Mini Plasmid Kit (TIANGEN, DP103-02). 1 × 105 HeLa cells were seeded in a well of 24-well plate. 24 h later, transient transfection was performed by following the manual of Lipofectamine™ 3000 Transfection Reagent (Invitrogen, L3000001). The total amount of plasmids in different wells were kept the same by adding pcDNA3.1-eGFP as the no-treatment control group and added pcDNA3.1-Dsup as the experimental group.

【3】After transfection of Dsup for 48 hours, take photos to record the cell cellular state or fluorescence intensity. At the same time, irradiate the cells with ultraviolet light for 15 s. Then placed them back to the CO2 incubator. Culture the cells for another 48 h. Then fix the cells by 4% PFA for 30 min.

【4】Observe the survival rate by using crystal violet staining methods and Image J software to calculate the intergrated density.

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# Team:QHFZ-China/Results

* [![Back to main page](https://static.igem.org/mediawiki/2020/3/36/T--QHFZ-China--%E9%98%9F%E5%BE%BD.jpg "Back to main page")](https://2020.igem.org/Team%3AQHFZ-China)
* [Project](https://2020.igem.org/Team%3AQHFZ-China/Description)
* [Model](https://2020.igem.org/Team%3AQHFZ-China/Model)
* [Human Practices](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
* [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
* [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* [Judging](https://igem.org/2020_Judging_Form?id=3457)
* [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)

* + [Description](https://2020.igem.org/Team%3AQHFZ-China/Description)
  + [Design](https://2020.igem.org/Team%3AQHFZ-China/Design)
  + [Proof Of Concept](https://2020.igem.org/Team%3AQHFZ-China/Proof_Of_Concept)
  + [Results](https://2020.igem.org/Team%3AQHFZ-China/Results)
  + [Implementation](https://2020.igem.org/Team%3AQHFZ-China/Implementation)
  + [Experiments](https://2020.igem.org/Team%3AQHFZ-China/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AQHFZ-China/Notebook)
* + [Modeling](https://2020.igem.org/Team%3AQHFZ-China/Model)
  + [Protein](https://2020.igem.org/Team%3AQHFZ-China/Protein)
* + [Intergrated](https://2020.igem.org/Team%3AQHFZ-China/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AQHFZ-China/Education)
* + [Parts](https://2020.igem.org/Team%3AQHFZ-China/Parts)
  + [Contribution](https://2020.igem.org/Team%3AQHFZ-China/Contribution)
  + [Engineering](https://2020.igem.org/Team%3AQHFZ-China/Engineering)
  + [Collection](https://2020.igem.org/Team%3AQHFZ-China/Part_Collection)
* + [Safety](https://2020.igem.org/Team%3AQHFZ-China/Safety)
* + [Judging](https://igem.org/2020_Judging_Form?id=3457)
* + [Team](https://2020.igem.org/Team%3AQHFZ-China/Team)
  + [Attributions](https://2020.igem.org/Team%3AQHFZ-China/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3AQHFZ-China/Collaborations)
  + [Partnership](https://2020.igem.org/Team%3AQHFZ-China/Partnership)
  + [Acknowledgement](https://2020.igem.org/Team%3AQHFZ-China/Acknowledgement)

[TOP](#top)

副页面

# Results

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[![tsinghua](https://static.igem.org/mediawiki/2020/1/10/T--QHFZ-China--tsinghua.jpg)](http://www.qhfz.edu.cn)
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