iGEM 2020 — Team XH-China

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

# Team:XH-China/Notebook

Document

![](https://static.igem.org/mediawiki/2020/5/56/T--XH-China--XH-Chinan_03.png)

KERATINASE SKIN EXFOLIATION

* [Home](https://2020.igem.org/Team%3AXH-China)
* [Attributions](https://2020.igem.org/Team%3AXH-China/Attributions)
* [Description](https://2020.igem.org/Team%3AXH-China/Description)
* [Contribution](https://2020.igem.org/Team%3AXH-China/Contribution)
* Project

  [Engineering](https://2020.igem.org/Team%3AXH-China/Engineering)
  [Experiments](https://2020.igem.org/Team%3AXH-China/Experiments)
  [Notebook](https://2020.igem.org/Team%3AXH-China/Notebook)
* [Collaborations](https://2020.igem.org/Team%3AXH-China/Collaborations)
* [Human-practices](https://2020.igem.org/Team%3AXH-China/Human_Practices)
* [Implementtation](https://2020.igem.org/Team%3AXH-China/Implementation)
* [Model](https://2020.igem.org/Team%3AXH-China/Model)
* [Entrepreneurship](https://2020.igem.org/Team%3AXH-China/Entrepreneurship)

### Notebook

### DAY1 7.31

After we got the plasmids, which was divided into two types. The first is carried by E. coli A, which is not good at expressing proteins. The second is a freeze-dried powder plasmid. Then we soak the second plasmid in saline, and we apply the second plasmid directly. Plasmids were placed in e. coli B and amplified on a medium plate. More importantly, we retained the first class plasmids embedded in E. coli A to prevent exhaustion.

### DAY2 8.4

The plasmid was extracted, and then double-enzyme digestion was followed by agar-gel electrophoresis (Nucleic acid electrophoresis). We need to test whether the plasmid that had been introduced into E. coli is containing the target gene, the experimental result was that the target gene was included. After three hours of IPTG induction, protein electrophoresis was carried out, but no protein was found to be expressed.

### DAY3 8.5

The reason is that the plasmids produced by the previous company did not contain his-tag and ampicillin resistant genes, which means that the vector plasmids containing the target genes sent by the company were not the vector plasmids that was required to be designed.So we're going to do a double digestion and move the target gene to a new plasmid that contains the HIS-tag and the ampicillin resistant gene.

### DAY4 8.6

Double enzyme digestion of plasmids containing the target gene, two bands to determine that the target gene has been cut, and then expansion of e. coli containing the vector plasmids.

![](https://static.igem.org/mediawiki/2020/2/2c/T--XH-China--Notebook_01.jpg)

### DAY 5 8.7

We extracted vector plasmids from the amplified Escherichia coli, configured with low melting point agar-gel electrophoresis, and used the cut plasmids containing the target gene to run the gel. After running the gel, the gel containing the target gene was directly cut and the target gene was recovered.

### DAY6 8.10

The target gene was recovered last Friday, but the band was very thin. After the plasmid concentration was measured, it was found that the plasmid concentration was low and could not be directly connected to the carrier plasmid.

### DAY7 8.11

Configure a low-melting agar-agar gel, run it with the target gene, then cut off the gel that contains the target gene, and this time recycle the target gene by putting it in a recycle column to increase its concentration.

![](https://static.igem.org/mediawiki/2020/c/c0/T--XH-China--Notebook_02.jpg)

### DAY8 8.12

Even so, the concentration of the target gene collected was still low, so this morning both the vector plasmid and the plasmid with the target gene were double-digested, paired with low melting point agarose gel, and given an increased dose in the card slot, using the two plasmids mentioned above. It turned out to be no good, double digestion did not cut the vector plasmid and plasmid with the target gene. Then, because the plasmid with the target gene had run out, the plasmid was extracted again.

![](https://static.igem.org/mediawiki/2020/2/2b/T--XH-China--Notebook_03.jpg)

### DAY9 8.13

Because the vector plasmid was also used up, we created the new competent cells (E. coli), the vector plasmid was converted, coated on a medium containing ampicillin, and one of the mediums was used as a negative control

### DAY10 8.14

Double enzyme digestion and nucleic acid electrophoresis were performed on the target plasmid extracted previously, but it was found that the cutting was not successful, because the cutting time was too short, so a single colony of bacteria was selected from the positive culture medium.

### DAY11 8.15

Ran electrophoresis of the target plasmid after the double enzyme digestion yesterday, found that the double enzyme digestion yesterday was successful, then extracted the vector plasmid amplified last night, and used the extracted vector plasmid to run gel in the afternoon, observed the concentration, and found that the concentration was high enough. At the same time, the target gene was cut into agar-agar gel with low melting point, and the target gene was directly cut into glue to recover. Then the target gene was connected with the extracted vector plasmid by double enzyme digestion.

![](https://static.igem.org/mediawiki/2020/e/e8/T--XH-China--Notebook_04.jpg)

### DAY12 8.18

The attached plasmids were transformed into E. coli and a number of single colonies were selected from the transformed plate and coated on the medium.

### DAY13 8.19

Single colonies were selected on the culture medium overnight.

### DAY14 8.20

The plasmid was extracted from the overnight shaking solution and verified by double enzyme digestion. Two bands were found, indicating that the target gene was successfully connected to the carrier plasmid. The plasmids were then extracted and transformed into DH5 (amplified E. coli), coated on culture medium, and transformed into Rosetta (e. coli for expression) and coated on culture medium.

### DAY15 8.21

Only one culture medium was found to have grown bacteria. However, because it was not labeled well, there was no way to tell whether the fungus on the culture medium was DH5 or Rosetta. So we first pick a single colony on the one with the bacteria and shake two tubes of bacteria.

### DAY16 8.22

One of the two tubes of shaken bacteria was added with inducer IPTG, and the other tube was left untreated. After centrifugation, the concentration of bacteria was increased, and then the two tubes of bacteria liquid were subjected to electrophoresis.

### DAY 17 8.24

After electrophoresis, it was found that the image was not displayed, so the bacteria were identified as DH5. After recoating Rosetta with medium, single colony shake bacteria were selected.

### DAY18 8.25

ITPG inducer was added to Rosetta overnight bacterial solution to induce expression, and the plasmid was extracted by run-protein electrophoresis three hours after induction.

### DAY19 8.26

The electrophoretic image showed that it was not expressed. The reason may be that ITPG was too old to use, so IPTG was reconfigured and the single colony was selected and put into the 1L culture bottle to cultivate Rosetta.

### DAY20 8.27

Inducers are added to induce Rosetta in a large culture flask and run to see if it expresses the protein.

### DAY21 8.28

Western blot of image signal is too weak, said there was no protein expression, so do the error analysis, plasmid extraction on Tuesday we came out and control plasmid DNA electrophoresis, found that we don't have image of plasmid, plasmid were not transformed into e. coli, e. coli but this is still in the AMP (ampicillin) of medium long out, even if not one of them contains AMP resistance genes. The reason may be due to AMP failure or other reasons, so a new tube of Rosetta bacteria that has been identified for plasmid introduction was found.

![](https://static.igem.org/mediawiki/2020/9/96/T--XH-China--Notebook_05.jpg)

### DAY22 8.29

The shaken Rosetta was poured into a 1L large culture flask, followed by IPTG induction for 3 hours, and protein electrophoresis was performed to determine whether the protein was successfully expressed. Fortunately, it worked.

![](https://static.igem.org/mediawiki/2020/7/70/T--XH-China--Notebook_06.jpg)

As shown in the figure, the expression of keratin in samples was induced by IPTG

and detected by SDS-PAGE and Western-blot for 3 h after induction.

Swimlane 1 is the electrophoretic band of the uninduced sample, and swimlane 2 is the induced expression sample band.Induced bands were detected in Wb test, and induced keratin could be expressed in resuspended bacterial fluid.

### DAY23 8.31

As shown in the figure, after inoculation of a large amount of keratin induced expression with the bacterial solution with a small amount of expression, the bacteria were collected by centrifugation, then ultrasonic crushing was performed, supernatant and precipitation were collected by centrifugation, and the precipitation was dissolved with 8M- urea buffer, and sampled and electrophoretic respectively.

Swimlane 1 is the electrophoresis band of supernatant sample, and swim lane 2 is the electrophoresis band of precipitate dissolved sample.

There were bands in both clearing and precipitation at the same size as keratin, so it was impossible to determine whether it was keratin or not, and western-blot detection was needed.

![](https://static.igem.org/mediawiki/2020/2/20/T--XH-China--Notebook_07.jpg)

### DAY24 9.1

As shown in the figure, after a large amount of expression, the bacteria were collected by centrifugation, followed by ultrasonic crushing, supernatant and precipitation were collected by centrifugation, and the precipitation was dissolved with 8M- urea buffer, respectively sampled and electrophoresis, and western-blot detection was carried out

Swimlane 1 is the electrophoresis band of supernatant sample, and swim lane 2 is the electrophoresis band of precipitate dissolved sample.

It can be seen from the figure that keratin is mainly expressed as soluble protein.

![](https://static.igem.org/mediawiki/2020/0/0c/T--XH-China--Notebook_08.jpg)

### DAY25 9.2

As shown in the figure, ultrasonic broken crude protein supernatant samples were taken for purification

Lane 1-3 is 20mM imidazole phosphate buffer to wash the mixed samples, adding in 5 times, each time adding 5mL.

Lane 4-7 is the sample eluted with 250mM imidazole phosphate buffer, which is added in 4 times and 5mL each time.

Keratin can be purified by ni-NTA preloaded with gravity, and the purified protein band is relatively single, the sample purity is high, but the yield is low.

![](https://static.igem.org/mediawiki/2020/6/61/T--XH-China--Notebook_09.jpg)

### DAY26 9.3

The activity of purified keratinase was measured by Sigma non-specific protease activity detection method, and the standard curve of content micromole and absorbance value OD660 was established. After calculation, the activity of the synthesized keratinase was calculated as 4.24 units /ml/ min.

Keratinase facial exfoliator

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

# Team:XH-China/Engineering

Document

![](https://static.igem.org/mediawiki/2020/5/56/T--XH-China--XH-Chinan_03.png)

KERATINASE SKIN EXFOLIATION

* [Home](https://2020.igem.org/Team%3AXH-China)
* [Attributions](https://2020.igem.org/Team%3AXH-China/Attributions)
* [Description](https://2020.igem.org/Team%3AXH-China/Description)
* [Contribution](https://2020.igem.org/Team%3AXH-China/Contribution)
* Project

  [Engineering](https://2020.igem.org/Team%3AXH-China/Engineering)
  [Experiments](https://2020.igem.org/Team%3AXH-China/Experiments)
  [Notebook](https://2020.igem.org/Team%3AXH-China/Notebook)
* [Collaborations](https://2020.igem.org/Team%3AXH-China/Collaborations)
* [Human-practices](https://2020.igem.org/Team%3AXH-China/Human_Practices)
* [Implementtation](https://2020.igem.org/Team%3AXH-China/Implementation)
* [Model](https://2020.igem.org/Team%3AXH-China/Model)
* [Entrepreneurship](https://2020.igem.org/Team%3AXH-China/Entrepreneurship)

### Engineering

During our implementation of our project, we are using synthetic biology and error analysis methods to make our experiment procedure concise, efficient, achieving engineering success, the processes of which are illustrated below.

![](https://static.igem.org/mediawiki/2020/9/98/T--XH-China--Engineering_01.png)

### Experiment design

A. Our group projected to auxotrophic bacteria, which requires extra organic growth factors and supplements on the culture medium. The auxotroph is unable to synthesize itself for its mutation. Therefore, using mutant bacteria enables us to regulate their enzyme synthesis by manipulating the availability of supplements.

Due to technology limits, unfortunately, the biology engineering company that we've been working with was incapable of constructing auxotroph with E.coli within two months.

B. We turned to an alternative plan. Yeast auxotroph was readily available in this company.

Above system till need different plasmids carrier, and applying auxotroph in the lab involves many uncertainties

C. It is better to use E.coli to achieve optimal results. We may add lactose operons or kill switches to attain the same effects.

![](https://static.igem.org/mediawiki/2020/9/98/T--XH-China--Engineering_01.png)

### During the experiment

### 1) At the beginning of the experiment：

A. We extracted the plasmid sent from the outsourcing company, and ran agar-gel electrophoresis (Nucleic acid electrophoresis) after the double enzyme digestion. In order to test whether the plasmid that had been introduced into E. coli contained the target gene, the experimental result was that it contained the target gene, and ran protein electrophoresis after induced by IPTG for three hours, but it was found that no protein was expressed.

We found that the reason is that the plasmid produced by the company before did not contain his-tag or Ampicillin resistance gene, that is to say, the carrier that the company sent was not the one we ordered.

B. Then double enzyme digestion was performed and separated to transfer the target gene to a new plasmid containing his-tag and ampicillin resistance genes.

### 2) When we tried to insert the target genes to the plasmid vector：

A. We extracted plasmids from cultured Escherichia coli without the target gene insertion, after digest with two restriction endonuclease BamHI/KpnI,the reaction sample was run electrophoresis with low melting point agar-gel to separate and purified the linear DNA fragment plasmid. Meantime, the plasmids containing the target gene also digest by above 2 endonuclease, then the reaction run electrophoresis to separate and purified target gene linear DNA fragment. After linear plasmid DNA and target gene DNA fragment harvest, the ligation of above two fragments had been performed.

The recovered target gene DNA fragment was used to run the agar-gel electrophoresis, and it was found that the band was very thin on the gel. Then the plasmid concentration was measured, and it was found that the plasmid concentration was low and could not be used to ligate with plasmid vector directly.

Then we collect more target gene DNA band from electrophoresis gel to increase the quantity of target gene DNA to meet the ligation need.

### 3)When we are using E.coli to produce keratinase protein：

A. We use E.coli DH5α to amplify plasmid and use E.coli Rossetta as protein expression host cell. Above E.coli could grow in culture medium with ampicillin because carrying plasmid with ampicillin resistance.

↓

B. Then a single colony picked on LB culture disk, inoculated in culture medium. When the bacteria grow to log growing phase (106CFU/ml), add inducer IPTG to one tube, and leave the other tube untreated. After induction 5-7 hours, harvest the bacteria and run electrophoresis on the SDS-PAGE gel. It was found that the target protein band showed on the gel with expected size comparing nothing show on SDS-PAGE gel loaded by sample without induction.

### 4)When we found that Rosetta with the inducer did not express keratin

A.Rosetta was induced in a large culture flask and run electrophoresis to confirm the protein produced in E.coli Rosetta by western blot .The band from western blot show not significant strong, which maybe caused by lower protein produce.

Error analysis. a.There maybe are some problem with transfection to E. coli; b. It may also caused by inducer ITPG deteriorates or fails.

B. We confirm the protein produced in E.coli with recombinant plasmid. We inoculate host bacteria in 1L large culture flask, IPTG induce 3-5 hours, harvest and purify target protein keratinase and tested.

Keratinase facial exfoliator

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

# Team:XH-China/Experiments

Document

![](https://static.igem.org/mediawiki/2020/5/56/T--XH-China--XH-Chinan_03.png)

KERATINASE SKIN EXFOLIATION

* [Home](https://2020.igem.org/Team%3AXH-China)
* [Attributions](https://2020.igem.org/Team%3AXH-China/Attributions)
* [Description](https://2020.igem.org/Team%3AXH-China/Description)
* [Contribution](https://2020.igem.org/Team%3AXH-China/Contribution)
* Project

  [Engineering](https://2020.igem.org/Team%3AXH-China/Engineering)
  [Experiments](https://2020.igem.org/Team%3AXH-China/Experiments)
  [Notebook](https://2020.igem.org/Team%3AXH-China/Notebook)
* [Collaborations](https://2020.igem.org/Team%3AXH-China/Collaborations)
* [Human-practices](https://2020.igem.org/Team%3AXH-China/Human_Practices)
* [Implementtation](https://2020.igem.org/Team%3AXH-China/Implementation)
* [Model](https://2020.igem.org/Team%3AXH-China/Model)
* [Entrepreneurship](https://2020.igem.org/Team%3AXH-China/Entrepreneurship)

### Summary of Experiments

### Why we pick Keratinase

At first Molyneux et al. (1959) attempted to isolate some bacteria that are able to degrade keratin. He isolated organisms from the contents of experimentally induced dermoid cysts from mid lateral region of sheep. Examination of wool sample showed degraded wool with numerous cortical and cuticular cells. He found disruption of wool fiber in both in vivo and in vitro. He showed that the organisms belong to genus Bacillus and the organism was capable of attacking native wool protein. The same year Noval et al. (1959) published another article on enzymatic decomposition of native keratin by Streptomyces fradiae. They showed extracellular enzyme secreted by these bacteria capable of degrading the human hair or other tissue in its native state. Many scientists did a lot of research work about keratinase and provide many interesting findings. They also notice that the enzyme-activity was inhibited by reduced glutathione, PMSF and 2-¬Mercaptaethanol..

According to above achievement and clue, we start our project with keratinase. We try to use this amazing bio-enzyme protein in beauty products. Bio-mask with keratinase to help degrade dead Keratinocyte and deep clean skin to make skin looks young, shiny and healthy. Therefore, the starting point and basis of the whole work is to obtain keratinase. As we all know, genetic engineering is an important method for protein synthesis. Therefore, using genetic engineering methods to prepare the keratinase we need is one of the main tasks of our laboratory.

**Plan of our experiment**

where did we work in?

Our laboratory located in beautiful Qianhai Free Trade Zone, Shenzhen, which belong to Shenzhen Blot Bio-products Ltd. There are very complete and advanced equipment and experienced technicians, which can fully guarantee the implementation of our project, and the teachers here are very friendly and professional, so any difficulties and problems encountered in our work can get their timely help and resolved.

![](https://static.igem.org/mediawiki/2020/b/ba/T--XH-China--exp01.jpg)

![](https://static.igem.org/mediawiki/2020/c/c2/T--XH-China--exp02.jpg)

![](https://static.igem.org/mediawiki/2020/3/31/T--XH-China--exp03.jpg)

![](https://static.igem.org/mediawiki/2020/e/e0/T--XH-China--exp04.jpg)

![](https://static.igem.org/mediawiki/2020/b/bc/T--XH-China--exp05.jpg)

![](https://static.igem.org/mediawiki/2020/a/a9/T--XH-China--exp06.jpg)

![](https://static.igem.org/mediawiki/2020/e/eb/T--XH-China--exp07.jpg)

![](https://static.igem.org/mediawiki/2020/f/fc/T--XH-China--exp08.jpg)

![](https://static.igem.org/mediawiki/2020/3/39/T--XH-China--exp09.jpg)

![](https://static.igem.org/mediawiki/2020/8/8d/T--XH-China--exp10.jpg)

![](https://static.igem.org/mediawiki/2020/2/2b/T--XH-China--exp11.jpg)

### Construction of Keratinase recombinant plamid

Before start our experiment, the first step is to search and select target keratinase gene. We use https://www.ncbi.nlm.nih.gov/ to search Keratinase gene, the search result show a few keratinase gene from different resource. Finally, we pick keratinase gene from Bacillus licheniformis. The gene sequence shows as following:

1 atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg

61 atggcattca gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat

121 attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag

181 agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac

241 aaagaagcgc ttaaggaagt caaaaatgat cccgatgtcg cttatgtgga agaggatcat

301 gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa

361 gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc

421 caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct

481 tataacaccg acggcaacgg acacggcaca catgttgccg gtacagtagc tgcgcttgac

541 aatacaacgg gtgtattagg cgttgcgcca agcgtatcct tgtacgcggt taaagtactg

601 aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca

661 aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa

721 caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc

781 ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct

841 gttggtgcgg tagactctaa cagcaacaga gcttcatttt ccagtgtggg agcagagctt

901 gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca

961 ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca

1021 aaacatccga acctttcagc ttcacaagtt cgcaaccgtc tctccagcac ggcgacttat

1081 ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaaggtgc cgctcaataa

Translation="MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGF

KSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVA

HALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEA

YNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWA

TTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYD

SVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGA

AALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ"

We chose this gene coding sequence as our target gene fragment for keratinase expression in E.coli. The main strategy is to insert the target gene sequence into an expression plasmid vector by genetic engineering to express keratinase. The technical pathway show as follows:

![](https://static.igem.org/mediawiki/2020/6/64/T--XH-China--Expriment_01.jpg)

The DNA synthesis order had been sent the commercial gene synthesis company to made keratinase gene sequence, which put in a regular plasmid with two specific restriction endonuclease BamHI/KpnI.

After we got received plasmid (powder and E.coli Top10 carry this plasmid), firstly we amplify this plasmid by growing above E.coli in LB medium, then isolate plasmid DNA from E.coli by plasmid extraction kit.

1.Digest the plasmid with restriction endonuclease BamHI/KpnI;

2.Confirm the expected size DNA insert by agarose gel electrophoresis;

3.After confirm the right insert DNA band is there, run above enzyme digest reaction on low melting argrose gel in TAE electrophoresis buffer;

4.Cut and purify the insert DNA band, the insert DNA had been purified by agarose DNA purify kit;

5.On the same time, plasmid pTrcHis2c also had been digest by endonuclease BamHI/KpnI and purify liner plasmid as above method;

6.Run ligation reaction above insert DNA and pTrcHis2c, then transfer ligation into E.coli host cell Rossete or BL21(DE3);

7.Inoculate above reaction on LB plate with Ampicillin(50ug/ml), 37℃, over night;

8.Pick single colony from above culture and confirm the recombinant plasmid is in host cell;

9.Inoculate the right colony in to 5ml LB broth (Ampicillin 50ug/ml), when the E. coli grow to 108/ml;

10.Inoculate above culture into 500ml LB broth, when shake in shaking incubator around 3-5hours (log grow phase), induce by adding IPTG, harvest the cell after 5 hours;

11.Check and confirm the recombinant keratinase expression by SDS-PAGE and western blot;

12.Harvest Keratinase by broken cell and Ni ion affinity chromatography to purify keratinase and test for following experiment.

### Evaluation of biological activity and safety of Keratinase

Use casein to detect the biological activity of artificial recombinant keratinase

A. Principle

Proteases can cleave peptide bonds. In the laboratory, it is usually necessary to measure or compare the activity of proteases. Sigma's non-specific protease activity test can be used as a standard method to measure protease activity. The test uses casein as the substrate. When the test protease is used to digest casein, tyrosine is released together with other amino acids and peptides. Folin & Ciocalteus phenol or Folin reagent can mainly react with free tyrosine and produce a blue chromophore, which can be quantified and measured by the absorbance value of a spectrophotometer. The more tyrosine released by casein, the more chromophores are produced, indicating that the protease activity is stronger. In the detection, the absorbance value produced by the protease activity is compared with the standard curve, and the standard curve is generated by reacting a known amount of tyrosine with FC reagent, and the change in absorbance value is compared with the amount of micromolar tyrosine Associated and generated. According to the standard curve, the activity of the protease sample can be determined in units, and the unit represents the micromolar amount of tyrosine equivalent released by casein per minute.

B. Material

Reagents:

Protease

Dipotassium hydrogen phosphate trihydrate

Casein

Trichloroacetic acid

Folin & Ciocalteu’s phenol reagent

Anhydrous sodium carbonate

Sodium acetate trihydrate

Calcium acetate

Free base L-tyrosine

equipment:

0.45 um polyether sulfone syringe filter and syringe

Dram sample bottle or polypropylene tube that can hold 15ml solution

Spectrophotometer

CuvettePipette

Stirring/heating plate

Stirring rod

Balance

pH meter

Drawing program

C. Reagent preparation

    1. 50mM potassium phosphate buffer, pH 7.5. During the preparation, 11.4 mg/ml of dipotassium hydrogen phosphate trihydrate was added to purified water, and the pH was adjusted with 1M HCl. Keep this solution at 37°C before use.

    2. Prepare a casein solution with a weight/volume ratio of 0.65% by mixing 6.5mg/ml of 50mM potassium phosphate buffer. Under gentle stirring, the temperature of the solution was gradually increased to 80-85°C and maintained for about 10 minutes until uniform dispersion was achieved. It must be ensured that boiling does not occur. Then use NaOH and HCl to adjust the pH as needed.

    3. 110mM trichloroacetic acid solution, prepared by diluting the 6.1N stock solution 1:55 with pure water. Trichloroacetic acid is a strong acid and should be handled with care.

    4. 0.5M Folin & Ciolcaltea’s or Folin phenol reagent can react with tyrosine and produce a measurable color change directly related to protease activity. Folin phenol reagent is an acid and needs to be handled with care.

    5. A 500mM sodium carbonate solution, prepared at 53mg/ml by adding anhydrous sodium carbonate to pure water.

    6. Enzyme dilution solution, composed of 10mM sodium acetate buffer (containing 5mM calcium, pH 7.5, 37°C). This solution can be used to dissolve solid protease samples or dilute enzyme solutions.

    7. 1.1 mM L-tyrosine standard stock solution. Add L-tyrosine to pure water to make a concentration of 0.2mg/ml, and slowly heat until tyrosine is dissolved. As with casein, no boiling occurs. After the L-tyrosine standard is cooled to room temperature, the solution can be further diluted to prepare a standard curve.

D. Establishment of standard curve

    1. Prepare 6 15ml polypropylene tubes, add 50ul, 100ul, 200ul, 400ul, 500ul 1.1mM tyrosine standard solution to the above-mentioned tubes, and set a blank control tube without adding tyrosine solution ；

    2. Fill the tube with pure water to make up the final volume to 2ml;

    3. Add 5ml 500mM sodium carbonate solution to the above tube, and immediately add 1ml Folin phenol reagent;

    4. Rotate and mix at 37°C for 30 minutes;

    5. Use a 0.45um filter to filter 2ml of the above solution for spectrophotometer detection;

    6. Detect the absorbance values of the above standard solution and blank solution at a wavelength of 660nm, and calculate the difference between the absorbance values of the standard solution and the blank solution;

    7. Take the change in absorbance of the standard as the Y axis, and use the corresponding micromolar amount of the 5 standard solutions as the X axis, and use the drawing program to draw the standard curve;

![](https://static.igem.org/mediawiki/2020/1/13/T--XH-China--Expriment_02.jpg)

    8. After we input the corresponding data, generate the standard curve and equation.

![](https://static.igem.org/mediawiki/2020/2/2b/T--XH-China--Expriment_10.jpg)

The fourth power polynomial regression equation is:

![](https://static.igem.org/mediawiki/2020/0/01/T--XH-China--Expriment_14.jpg)

Where:

p1 = 5.423

p2 = -11.43

p3 = 8.855

p4 = -1.8

p5 = 0.1558

E. Protease activity detection

1. Take 4 15ml polypropylene tubes, one of which will be used as a blank control, and the other three will be used for three different dilutions of keratinase;

2. Add 5ml of 0.65% casein solution to the above four sample bottles, and let them stabilize in a water bath at 37°C for about 5 minutes;

3. Add 20ul, 50ul and 100ul keratinase solution to the three test sample bottles other than the blank control bottle. Rotate to mix and incubate at 37°C for 10 minutes;

4. After incubating for 10 minutes, add 5ml trichloroacetic acid TCA reagent to each tube to stop the reaction. Then add an appropriate amount of enzyme solution to the above-mentioned tubes including the blank tube so that the final volume of the enzyme solution in each tube is 0.1 ml;

5. Incubate the above solution at 37°C for 30 minutes;

6. After incubating for 30 minutes, use a 0.45um filter to filter each 2ml of the above solution and transfer it to a new polypropylene tube;

7. Add 5ml 500mM sodium carbonate to the above tube, and immediately add 1ml Folin phenol reagent;
8. Rotate the above solution to mix and incubate at 37°C for 30 minutes;

9. Use a 0.45um filter to filter 2ml of the above solution until colorimetric detection and record the corresponding absorbance value.

Result：

Table 2. Spectrophotometer test results after keratinase digested casein

![](https://static.igem.org/mediawiki/2020/6/6a/T--XH-China--Expriment_12.jpg)

E. Measure absorbance and calculate enzyme activity

Then, we can substitute the difference in absorbance between each test keratinase and the blank sample into the slope formula to calculate the corresponding amount of tyrosine (uMol) in this specific proteolysis reaction. Subsequently, the corresponding enzyme activity value in units/ml is obtained based on the following formula.

![](https://static.igem.org/mediawiki/2020/f/ff/T--XH-China--Expriment_05.jpg)

11= The total volume of the detection reaction (ml)

10= Detection time per unit (minutes)

0.1= Volume of enzyme used (ml)

2= Volume used in colorimetric measurement (ml)

The number of micromoles of tyrosine divided by the number of minutes of time is the unit value of protease activity we measured.

Result:

Table 4. Keratinase activity

![](https://static.igem.org/mediawiki/2020/1/10/T--XH-China--Expriment_13.jpg)

F. Conclusion

Therefore, the activity of the synthesized keratinase is 4.24 units/ml/min

### Function evaluation of Bio-mask with Keratinase

Prepare cream mask base. (Bought from Shanghai woyou media Ltd. Total 500g);

![](https://static.igem.org/mediawiki/2020/3/36/T--XH-China--Expriment_07.jpg)

2.Prepare Keratinase protein solution 300ug/ml;

3.Mix keratinase solution with cream mask base to make biological cream mask with different concentration keratinase, such as 10ug/ml,5ug/ml,0.5ug/ml and 0.1ug/ml;

![](https://static.igem.org/mediawiki/2020/5/5a/T--XH-China--Expriment_08.jpg)

4.The highest keratinase concentration 10ug/ml cream mask had been used to test Bio mask’s sfety;

Results showed as below:

![](https://static.igem.org/mediawiki/2020/f/f5/T--XH-China--Expriment_09.jpg)

Conclution:

The biological mask with keratinase can clean the skin very well, make the skin moisturized, youthful, and full of elasticity, change the appearance of the skin without any harm to the skin.

Keratinase facial exfoliator

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

# Team:XH-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.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)