iGEM 2021 — Team QHFZ

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# Team:QHFZ/Notebook

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# 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.

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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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# Team:QHFZ/Engineering

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* [Team](https://2021.igem.org/Team%3AQHFZ/Team)

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  + [Partnership](https://2021.igem.org/Team%3AQHFZ/Partnership)
  + [Acknowledgement](https://2021.igem.org/Team%3AQHFZ/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://2021.igem.org/Team%3AQHFZ/Description) and [Proof Of Concept](https://2021.igem.org/Team%3AQHFZ/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.

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# Team:QHFZ/Experiments

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  + [Acknowledgement](https://2021.igem.org/Team%3AQHFZ/Acknowledgement)

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# Experiments

## Experimental considerations

1、Configurate culture medium
    Because AGAR is insoluble in medium at low temperature, it can not be mixed with a large bottle and then packaged, and then directly autoclave sterilization;

High temperature will make antibiotics ineffective, so antibiotics must be added after sterilization, after the medium is slightly cold, about the temperature is not hot back, pay attention to not too cold, otherwise it will solidify;

    After the medium is solidified, cover with plastic film and store at 4℃. All reagents should be returned (especially antibiotics, put back to -20℃).

2、 Need to configurate your own reagent
    Some (e.g., chloramphenicol) are dissolved in a solvent other than water;

    When using analytical balance to accurately weigh, because it is not easy to accurately control the weight, so you can first weigh the solid, calculate the required liquid volume configuration solution;

    Some reagents need to be filtered by bacterial membrane.

3、Sterilization
    Single layer newspaper packaging of gun head, very wet after quenching, put in the oven to dry out; Conical bottles, etc., are packed with sealing film and double layers of newspaper, and the rubber band is tightened to the bottom of the bottle mouth (there can be more sterile area);

    The sugar-containing medium was sterilized at 115℃, and the rest at 121℃;

    Before use, check the water level inside and outside the pot, whether the water at the bottom of the pot is not through the hole, whether the water in the bucket outside the pot is between the highest and lowest line, if you want to fill water only distilled water.

    Cover abandoned culture medium with newspaper to prevent overflow when using a leakproof bucket

    Don't tighten anything with a lid, like a glycerine tube

    Those that need to be sterilized in a leakproof iron bucket: one that feels liquid flowing out (e.g. glass petri dish) and another that is heated and deformed (plastic petri dish, 96-well plate).

    Those that need to be sterilized in common frame: head box, conical bottle, lunch box, etc.

4、the operation of inverted plate, inoculation, marking and coating
    When you put the culture medium into the incubator, don’t forget to take it out on time.

    Sometimes the sealing film plate will also be dried, if a long time to cultivate remember to choose a less windy location

5、Thermostatic cultivation

Always open something from the shaker, someone else may still be using it

    Remember to turn on the cooling switch

6、Centrifuge
    Be sure to balance it

    Cover it when you're done!

7、PCR
    Change the tip at any time, do not contaminate the reagent!

    Enzyme dissolved on ice, do not hand cover, use immediately put back to -20℃.

    After using the enzyme, put it into the centrifuge will come out a few μL, be sure to use it properly.

    If it is colony PCR, a large mixture system can be made with a large number of samples and then packaged

    If the extension time difference is not very large, several different things can be PCR at the same time. The time is matches with the longest one, and the annealing temperature is matches with the lowest one.

    Gel can start prepared when PCR has more than 30 minutes left

8、Agarose gel electrophoresis
    Use Agarose gel electrophoresis with different concentration for different length segments, remember to read the instructions ！！！！

    Dissolve the AGAR in the microwave until the solution is completely clear. Do not boil for too long to evaporate the solution

    Goldview/dye is added after the solution cools slightly to prevent thermal denaturation.

    As you pull out the comb, pull it vertically bit by bit, remove the gel and scrape the bottom.

    The Running buffer should be submerged over the gel.

    Sample loading as soon as possible to prevent the first sample floating out.

9、In-fusion cloning
    Calculate the proportion of each fragment according to the corresponding protocol. If the fragment is long, the time of 50℃ can be appropriately extended (see the instructions for details).

    Resuscitation can be incubated for 30 minutes before adding the corresponding antibiotics

10、Plasmid extraction (using kit)
    Before adding solution I, re-suspend all bacteria in a tube with the bacteria solution. Solution I stored at 4℃

    Solution III requires precooling

    Every time after pouring the liquid, tap the tube vigorously. If the liquid is not poured clean, the filtered membrane will contact the filtrate below during the final centrifugation, which is equivalent to white centrifugation

    Tick the bottle after using Wb to add ethanol and check to see if it has been added before using WB

    Preheat EB/ddH2O in advance during elution (ddH2O elution must be used if sequencing is to be sent (ddH2O elution can also be used without sequencing)

    Open the cover to evaporate the alcohol before adding the eluent into the column. When adding the eluent, drop it into the center of the filter membrane, cover the cover and continue to heat for 1-2 minutes

    The elution volume of plasmid and gel is different! Look at the instructions

    Heat elution and cold centrifugation

11、Slice&recycle the gel (using kit)
    When GM is dissolved, it can be heated moderately and oscillated until the liquid flows smoothly, does not appear oily and does not stick to the bottom of the tube.

    Be sure to cover the tube tightly when shaking, because GM is corrosive.

12、Make glycerine tube
    Use reverse pipetting to remove glycerol - this method is used for thick liquids

    The glycerol tube should be marked, and the label should be firmly attached (it will fall off after being thawed several times). It can also be marked before sterilization, and the thermal movement of molecules can make the mark not easy to disappear.

## Experimental Skill Training

Several highlighted issues:

    1） Non-contaminated area, contaminated area must pay attention to, strictly prohibit confusion. (Contaminated area: run glue area, all areas that will come into contact with dye, PCR machine, refrigerator are non-contaminated area!)

    2） The pipette gun and medicine should be put back in place

    3） The centrifuge cover must be tightened and closed

    4） After use, the shaker must be reopened if there is anything in it

    5） Discard the gel immediately after use

    6） The experimental area and living area are clear

    7） Be sure to put the enzyme back in the refrigerator after using it

    8） Always tell someone when you're moving hisvor her stuff

    All procedures should follow the protocol on the purchased kit!!

# 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/Results

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* [Team](https://2021.igem.org/Team%3AQHFZ/Team)

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* + [Judging](https://2021.igem.org/Team%3AQHFZ/Judging)
* + [Team](https://2021.igem.org/Team%3AQHFZ/Team)
  + [Attributions](https://2021.igem.org/Team%3AQHFZ/Attributions)
  + [Collaboration](https://2021.igem.org/Team%3AQHFZ/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AQHFZ/Partnership)
  + [Acknowledgement](https://2021.igem.org/Team%3AQHFZ/Acknowledgement)

[TOP](#top)

# 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)