iGEM 2021 — Team GDSYZX

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# Team:GDSYZX/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:GDSYZX/Engineering

[GDSYZX](https://2021.igem.org/Team%3AGDSYZX)

* [HOME](https://2021.igem.org/Team%3AGDSYZX)
* PROJECT
  + [Description](https://2021.igem.org/Team%3AGDSYZX/Description)
  + [Results](https://2021.igem.org/Team%3AGDSYZX/Results)
  + [Contribution](https://2021.igem.org/Team%3AGDSYZX/Contribution)
  + [Excellence in Another Area](https://2021.igem.org/Team%3AGDSYZX/Excellence_in_Another_Area)
* [MODEL](https://2021.igem.org/Team%3AGDSYZX/Model)
* [SOFTWARE](https://2021.igem.org/Team%3AGDSYZX/Software)
* [IMPROVED PARTS](https://2021.igem.org/Team%3AGDSYZX/Improved_Parts)
* EXPERIMENT
  + [Proof of concept](https://2021.igem.org/Team%3AGDSYZX/Proof_Of_Concept)
  + [Implementation](https://2021.igem.org/Team%3AGDSYZX/Implementation)
  + [Engineering](https://2021.igem.org/Team%3AGDSYZX/Engineering)
  + [Design](https://2021.igem.org/Team%3AGDSYZX/Design)
  + [Build](https://2021.igem.org/Team%3AGDSYZX/Build)
  + [Test](https://2021.igem.org/Team%3AGDSYZX/Test)
  + [Parts](https://2021.igem.org/Team%3AGDSYZX/Parts)
* HP
  + [Human Practices](https://2021.igem.org/Team%3AGDSYZX/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AGDSYZX/Collaborations)
  + [Communication](https://2021.igem.org/Team%3AGDSYZX/Communication)
  + [Education](https://2021.igem.org/Team%3AGDSYZX/Education)
  + [Safety](https://2021.igem.org/Team%3AGDSYZX/Safety)
* TEAM
  + [Attributions](https://2021.igem.org/Team%3AGDSYZX/Attributions)
  + [Members](https://2021.igem.org/Team%3AGDSYZX/Members)
* [PROTOCAL](https://2021.igem.org/Team%3AGDSYZX/protocal)

* [Project Background Investigation](#section1)
* [STEP 1: Analysis of RNA- Seq data](#section2)
* [STEP 2: Selection of candidate promoters](#section3)
* [STEP 3: Screening of efficient feather-degrading strains](#section4)
* [Conclusion](#section5)
* [Further Research Directions](#section6)

![](https://static.igem.org/mediawiki/2021/c/cc/T--GDSYZX--experiement.jpg)

# ENGINEERING

## Project Background Investigation

For more information and references, please see our PROJECT DESCRIPTION.

(1) The conditions of feather waste generated by poultry industry.

(2) The current industrial treatment of feathers.

(3) The most promising alternative approaches involve biodegradation using keratinases or bacteria.

(4) The feather degradation mechanisms of Streptomyces sp. SCUT-3.

(5) Sep39 and CDO1 are the key enzyme involved in the hydrolysis of feather and co-overexpression can increase the keratinase activity of SCUT-3 but yield fewer cells.

(6) To further improve the degradation efficiency without influencing the growth rate, we attempt to modify SCUT-3.

## STEP 1: Analysis of RNA- Seq data

Research → Imagine → Design → Test → Learn → Improve → Research...

(1) Research

We can find appropriate promoters to replace *ermE* promoter through analyzing RNA-Seq datas from wild type SCUT-3 cultured on feather and chitin.

(2) Imagine

The appropriate promoters for overexpression of *Sep39* and *CDO1* can be found in RNA-Seq datas.

[https://2021.igem.org/Team:GDSYZX/Build](https://2021.igem.org/Team%3AGDSYZX/Build)

(3) Design

We cultured wild type SCUT-3 on feather and chitin, extracted their RNA and sent their RNA to company for RNA-Seq analysis.

[https://2021.igem.org/Team:GDSYZX/Design](https://2021.igem.org/Team%3AGDSYZX/Design)

(4) Test

Based on RNA-Seq data, using TPM as the gene expression intensity evaluation index, 9 candidate promoters were selected in total.

[https://2021.igem.org/Team:GDSYZX/Test](https://2021.igem.org/Team%3AGDSYZX/Test)

(5) Learn

RNA-Seq data results showed that the selected promoters could be used in the following experiment.

[https://2021.igem.org/Team:GDSYZX/Results](https://2021.igem.org/Team%3AGDSYZX/Results)

(6) Improve

Confirm whether the selected promoters can actually replace *ermE* promoter.

## STEP 2: Selection of candidate promoters

→ Imagine → Design →Test → Learn → Research...

(1) Imagine

The selected promoters can actually replace *ermE* promoter.

(2) Design

To verify the actual expression strength of the selected promoters, we can chose enhanced green fluorescent protein eGFP as the reporter gene.We could replace the *ermE* promoter located on the Streptomyces integrative plasmid *pSET152* with the promoters cloned and obtaine the recombinant *pSET152* vector carrying different promoters. Promoters that exhibited higher or lower fluorescence intensity than *ermE* promoter are chosen.

[https://2021.igem.org/Team:GDSYZX/Build](https://2021.igem.org/Team%3AGDSYZX/Build)

(3) Test

The fluorescence intensity of SCUT-3 integrated with different *pSET152-promoter-eGFP* recombinant expression vector were detected.

[https://2021.igem.org/Team:GDSYZX/Test](https://2021.igem.org/Team%3AGDSYZX/Test)

(4) Learn

We found that among all the 8 screened promoters, the fluorescence intensity of *pro1380* and *pro24880* were significantly higher than *ermE*, and the the fluorescence intensity of *pro22610, pro15290, pro15290* were lower than *ermE*. We choosed *pro24880, pro22610* and *pro1380* as our candidate promoters.

[https://2021.igem.org/Team:GDSYZX/Results](https://2021.igem.org/Team%3AGDSYZX/Results)

## STEP 3: Screening of efficient feather-degrading strains

→ Imagine → Design →Test → Learn → Research...

(1) Imagine

To increase the degradation efficiency of SCUT-3 and not affect the growth of SCUT-3, we constructed recombinant Streptomyces by using promoters we selected to replace *ermE*. Later we can teste their feather degradation efficiency and the bacterial growth condition.

(2) Design

①Construction of recombinant Streptomyces. To obtain stronger industrial feather-degrading strains, we decided to genetically modifying these SCUT-O*cdo-*O*sep39* strains by replacing promoter *ermE* with the promoters we have chosen. ②Screening of efficient feather-degrading strains. To verify our hypothesis, the feather degradation efficiency of strain was compared with wild-type SCUT-3 and SCUT-O*cdo-*O*sep39*. ③Testing the bacterial growth condition.

[https://2021.igem.org/Team:GDSYZX/Design](https://2021.igem.org/Team%3AGDSYZX/Design)

(3) Test

The feather degradation efficiency and bacterial growth condition of recombinant strain was tested and compared with wild-type SCUT-3 and SCUT-O*cdo-*O*sep39*.

[https://2021.igem.org/Team:GDSYZX/Test](https://2021.igem.org/Team%3AGDSYZX/Test)

(4) Learn

The feather degradation efficiency of recombinant strain SCUT-O*cdo*-p24880-O*sep39* was higher than wild-type SCUT-3 and SCUT-O*cdo-*O*sep39* on 48h. The bacterial growth condition tests showed that the overexpression of *sep39* under promoter *p24880* did not affect wild type scut’s growth.

[https://2021.igem.org/Team:GDSYZX/Results](https://2021.igem.org/Team%3AGDSYZX/Results)

## Conclusion

We successfully that the SCUT-O*cdo*-p24880-*Osep39* strain showed stronger keratinase activity, higher production of peptides and amino acids and great changes in feather degradation rate. The bacterial growth condition tests showed that the SCUT-O*cdo*-p24880-O*sep39* strain grew similarly to the wild type, both were able to form clearly visible and large colonies, much better than the group with constitutive promoter.

[https://2021.igem.org/Team:GDSYZX/Results](https://2021.igem.org/Team%3AGDSYZX/Results)

## Further Research Directions

Because of the resulting pollution, amino acid destruction, low solubility and difficult to be digested by animals, traditional physical/chemical treatment methods for feather waste are gradually being abandoned [1]. The most promising alternative approaches involve biodegradation using bacteria. Through studies of using microbial means to convert accumulated millions tonnes of feathers into keratin, we take one step further to accomplish turning feather, instead of a burdensome waste, into rich, widely usable resource of protein and valuable raw materials for many fields.

(1) Biomedical applications of keratin. Keratin based biomaterials have been widely produced and used in various biomedical applications, such as bone tissue engineering[2, 3], wound healing[4, 5], nerve regeneration[6], skin replacement[7] and controlled drug delivery[8-10]. Our research is a simple, green, cost effective and yet efficient methodologies for the better extraction of keratin from feather. It is expected that keratin we extracted will turn into a mainstream biomaterial for clinical trials.

(2) Feather is a natural source of amino acids, peptides, and minerals that can be used in animal feed supplements and as a nitrogen fertilizer for plants[11]. Biodegradation of feather by microbial is a safe, sanitary and cost-effective technology that can play an important role in in processing feather waste.

Our study would shed light on the feather utilization mechanisms of FDB and provide a highly efficient, cost-effective way for degradation of feathers and extraction of available keratin, amino acids. Further exploration of the physiological functions of *CDO1* and *Sep39* in this Streptomyces. SCUT-3 will likely provide additional insights into biodegradation using bacteria.

## Reference

[1] Li Z W, Liang S, Ke Y, et al. The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3 [J]. Commun Biol, 2020, 3(1): 191.

[2] Li J S, Li Y, Liu X, et al. Strategy to introduce an hydroxyapatite-keratin nanocomposite into a fibrous membrane for bone tissue engineering [J]. J Mater Chem B, 2013, 1(4): 432-7.

[3] Tachibana A, Nishikawa Y, Nishino M, et al. Modified keratin sponge: Binding of bone morphogenetic protein-2 and osteoblast differentiation [J]. Journal of Bioscience and Bioengineering, 2006, 102(5): 425-9.

[4] Ham T R, Lee R T, Han S, et al. Tunable keratin hydrogels for controlled erosion and growth factor delivery [J]. Biomacromolecules, 2016, 17(1): 225-36.

[5] Nakata R, Tachibana A, Tanabe T. Preparation of keratin hydrogel/hydroxyapatite composite and its evaluation as a controlled drug release carrier [J]. Mater Sci Eng C Mater Biol Appl, 2014, 41(59-64.

[6] Lin Y C, Ramadan M, Van Dyke M, et al. Keratin gel filler for peripheral nerve repair in a rodent sciatic nerve injury model [J]. Plast Reconstr Surg, 2012, 129(1): 67-78.

[7] Xu S, Sang L, Zhang Y, et al. Biological evaluation of human hair keratin scaffolds for skin wound repair and regeneration [J]. Mater Sci Eng C Mater Biol Appl, 2013, 33(2): 648-55.

[8] Yin X C, Li F Y, He Y F, et al. Study on effective extraction of chicken feather keratins and their films for controlling drug release [J]. Biomater Sci, 2013, 1(5): 528-36.

[9] Guo J, Pan S, Yin X, et al. pH-sensitive keratin-based polymer hydrogel and its controllable drug-release behavior [J]. Journal of Applied Polymer Science, 2014.

[10] Shavandi A, SilVA T H, Bekhit A A, et al. Keratin: dissolution, extraction and biomedical application [J]. Biomater Sci, 2017, 5(9): 1699-735.

[11] Kornillowicz-kowalska T, Bohacz J. Biodegradation of keratin waste: Theory and practical aspects [J]. Waste Manag, 2011, 31(8): 1689-701.

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# Team:GDSYZX/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:GDSYZX/Results

[GDSYZX](https://2021.igem.org/Team%3AGDSYZX)

* [HOME](https://2021.igem.org/Team%3AGDSYZX)
* PROJECT
  + [Description](https://2021.igem.org/Team%3AGDSYZX/Description)
  + [Results](https://2021.igem.org/Team%3AGDSYZX/Results)
  + [Contribution](https://2021.igem.org/Team%3AGDSYZX/Contribution)
  + [Excellence in Another Area](https://2021.igem.org/Team%3AGDSYZX/Excellence_in_Another_Area)
* [MODEL](https://2021.igem.org/Team%3AGDSYZX/Model)
* [SOFTWARE](https://2021.igem.org/Team%3AGDSYZX/Software)
* [IMPROVED PARTS](https://2021.igem.org/Team%3AGDSYZX/Improved_Parts)
* EXPERIMENT
  + [Proof of concept](https://2021.igem.org/Team%3AGDSYZX/Proof_Of_Concept)
  + [Implementation](https://2021.igem.org/Team%3AGDSYZX/Implementation)
  + [Engineering](https://2021.igem.org/Team%3AGDSYZX/Engineering)
  + [Design](https://2021.igem.org/Team%3AGDSYZX/Design)
  + [Build](https://2021.igem.org/Team%3AGDSYZX/Build)
  + [Test](https://2021.igem.org/Team%3AGDSYZX/Test)
  + [Parts](https://2021.igem.org/Team%3AGDSYZX/Parts)
* HP
  + [Human Practices](https://2021.igem.org/Team%3AGDSYZX/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3AGDSYZX/Collaborations)
  + [Communication](https://2021.igem.org/Team%3AGDSYZX/Communication)
  + [Education](https://2021.igem.org/Team%3AGDSYZX/Education)
  + [Safety](https://2021.igem.org/Team%3AGDSYZX/Safety)
* TEAM
  + [Attributions](https://2021.igem.org/Team%3AGDSYZX/Attributions)
  + [Members](https://2021.igem.org/Team%3AGDSYZX/Members)
* [PROTOCAL](https://2021.igem.org/Team%3AGDSYZX/protocal)

* [1. Analysis of RNA- Seq data](#section1)
* [2. Selection of candidate promoters](#section2)
* [3. Construction of recombinant Streptomyces](#section3)
* [4. Screening of efficient feather-degrading strains](#section4)
  + [4.1 Determination of keratinase activities](#section41)
  + [4.2 Determination of amino acid content](#section41)
  + [4.3 Determination of peptide content](#section42)
  + [4.4 Determination of peptide and amino acid content](#section43)
  + [4.5 Analysis of feather degradation rate](#section44)
* [5. Growth conditions of various SCUT-3 strains](#section5)
* [Future Prospects and Concern](#section6)
* [References](#section7)

![](https://static.igem.org/mediawiki/2021/7/70/T--GDSYZX--project.JPG)

# RESULTS

## 1.Analysis of RNA- Seq data

In order to find appropriate promoters, we obtained RNA-Seq datas from wild type SCUT-3 cultured on feather and chitin. Based on RNA-Seq data, using TPM as the gene expression intensity evaluation index, 9 candidate promoters were selected in total (Figure 1). These nine promoters are *pro1380, pro2953, pro3035, pro3040, pro3071, pro05270, pro15290, pro22610* and *pro24880*. The lengths of these promoters are shown in Table 1.

![](https://static.igem.org/mediawiki/2021/3/36/T--GDSYZX--Results-fig1.png)

Figure 1. Expression strength of promoters. A: promoters selected from SCUT-3 cultured on feather; B: promoters selected from SCUT-3 cultured on chitin.

Table 1 The name and length of candidate promoters.

| Promter name | Promoter length (bp) |
| --- | --- |
| *pro1380* | 300 |
| *pro2953* | 303 |
| *pro3035* | 403 |
| *pro3040* | 263 |
| *pro3071* | 193 |
| *pro15290* | 218 |
| *pro22610* | 152 |
| *pro24880* | 461 |

## 2. Selection of candidate promoters

By using the genome of SCUT-3 as template, the candidate promoters were amplified by PCR. We successfully cloned 8 kinds promoters, including *pro1380, pro2953, pro3035, pro3040, pro3071, pro05270, pro15290, pro22610* and *pro24880*. The PCR results of each promoter are shown in Figure 2.

![](https://static.igem.org/mediawiki/2021/7/7d/T--GDSYZX--Results-fig2.png)

Figure 2. The colony PCR results of each *promoter-eGFP* recombinant expression vector. A: The colony PCR results of*pro1380/ pro2953/ pro3035/ pro3040/ pro3071-eGFP* and recombinant expression vector. Lane 1: DNA marker. Lane 2~5：*pro1380*. Lane 6~9：*pro2953*. Lane 10~12：*pro3035*. Lane 13~14：*pro3040*. Lane 15~16：*pro3071*. B: The colony PCR results of *pro15290-eGFP* and recombinant expression vector. Lane 1: DNA marker. Lane 2~11：*pro15290*. C: The colony PCR results of *pro24880-eGFP* and recombinant expression vector. Lane 1: DNA marker. Lane 2~11：*pro24880*. D: The colony PCR results of *pro22610-eGFP* and recombinant expression vector. Lane 1: DNA marker. Lane 2~11：*pro22610*.

To verify the actual expression strength of the selected promoters, we chose enhanced green fluorescent protein eGFP as the reporter gene. After replacing the *ermE* promoter located on the Streptomyces integrative plasmid *pSET152* with the promoters cloned, we obtained the recombinant *pSET152* vector carrying different promoters. The colony PCR results are shown in Figure 3.

![](https://static.igem.org/mediawiki/2021/d/d4/T--GDSYZX--Results-fig3.png)

Figure 3. The colony PCR results of each *promoter-eGFP* recombinant expression vector. A: Lane 1: DNA marker. Lane 2~5：pro *ermE*. B: Lane 6: DNA marker. Lane 1~5：*pro1380*. Lane 7~11：*pro3035*. C: Lane 6: DNA marker. Lane 1~5：*pro3040*. Lane 7~11：*pro22610*. D: Lane 6: DNA marker. Lane 1~5：*pro15290*. Lane 7~11：*pro22610*. E: Lane 6: DNA marker. Lane 1~5：*pro24880*.

*pSET152* is an integrated plasmid. After the recombinant *pSET152* vector was introduced into SCUT-3, the plasmid will be integrated into the genome of SCUT-3. Therefore, we can design primers with the plasmid base sequence to identify positive conjugant. After identification, 8 *promoters-eGFP* expression vectors were successfully integrated into the SCUT-3 genome respectively, as shown in Figure 4.

![](https://static.igem.org/mediawiki/2021/a/ad/T--GDSYZX--Results-fig4.png)

Figure 4. The colony PCR results of SCUT-3 genome integrated with *pSET152-promoter-eGFP* vector. A: Lane 1: DNA marker. Lane 2：SCUT-3. Lane 3~4：SCUT-3 genome integrated with *pSET152-ermE-eGFP* vector. B: Lane 1: DNA marker. Lane 2~8：SCUT-3 genome integrated with *pSET152-pro1380/pro3035/pro3040/pro3071/pro15290/pro22610/pro24880-eGFP* vector respectively. Lane 10: SCUT-3.

We detected the fluorescence intensity of SCUT-3 integrated with different *pSET152-promoter-eGFP* recombinant expression vector. We found that among all the 8 screened promoters, the fluorescence intensity of *pro1380* and *pro24880* were significantly higher than ermE, and the the fluorescence intensity of *pro3040, pro15290, pro15290* were lower than ermE, as shown in Figure 5.

![](https://static.igem.org/mediawiki/2021/9/9e/T--GDSYZX--Results-fig5.png)

Figure 5. Comparison of *eGFP* fluorescence intensity driven by different promoters.

## 3. Construction of recombinant Streptomyces

In our previous work, we found that co-overexpression of *CDO1* and *SEP39* (both used constitutive promoter *ermE*, which was named SCUT-O*cdo-*O*sep39*, increased SCUT-3’s feather degrading efficiency but SCUT-O*cdo-*O*sep39* showed lower bacterial cell copy number than wild-type SCUT-3. To obtain stronger industrial feather-degrading strains, we decided to genetically modifying these SCUT-O*cdo-*O*sep39* strains by replacing promoter *ermE* with the promoters we have chosen. Fortunately, we successfully constructed SCUT-O*cdo-*p1380-O*sep39*, SCUT-p22610-O*cdo-*O*sep39* and SCUT-O*cdo-*p24880-O*sep39* strains by using promoter *pro1380, pro22610, pro24880* respectively. The colony PCR results are shown in Figure 6.

![](https://static.igem.org/mediawiki/2021/d/d6/T--GDSYZX--Results-fig6.png)

Figure 6. .The colony PCR results of recombinant Streptomyces genome promoter. Lane 1: DNA marker. Lane 2~3：The colony PCR results of *pro1380*. Lane 4~5：The colony PCR results of *pro22610*. Lane 6~7：The colony PCR results of *pro24880*.

## 4. Screening of efficient feather-degrading strains

Feather degradation by FDB (Feather degrading bacteria) is completed by the integration of two processes, disulfide bond reduction and protease hydrolysis. CDO is a non-heme mononuclear iron enzyme that oxidizes cysteine to cysteine sulfinic acid, which is then dissimilated by aminotransferase into pyruvate and sulfite. In SCUT-3’s feather degradation, overexpression of CDO1 increased SCUT-3’s single-cell feather degradation efficiency. SEP39 is a major protease involved in SCUT-3’s keratin hydrolysis, and previous work showed that its overexpression could increase SCUT-O*sep39*’s keratinase activity. We previously showed that co-overexpression of *CDO1* and *SEP39 by* using constitutive promoter *ermE* increased SCUT-3’s feather degrading efficiency, which implied that *CDO* and *SEP39* play an important role in the degradation of feather. However, we also found that SCUT-O*cdo-Osep39* showed lower growth rate than wild-type SCUT-3. To increase the degradation efficiency of SCUT-3 and not affect the growth of SCUT-3, we constructed recombinant Streptomyces by using promoters we select to replace *ermE*.

To verify our hypothesis, the feather degradation efficiency of strain SCUT-O*cdo-*p1380*-sep39*, SCUT-p22610-O*cdo*-O*sep39* and SCUT-O*cdo*-p24880-O*sep39* was compared with wild-type SCUT-3 and SCUT-O*cdo*-O*sep39*. The total peptide and amino acid yields of were highest in SCUT-O*cdo-*p24880-O*sep39*, and tests indicated that there was significant difference between SCUT-O*cdo-*p24880-O*sep39* and SCUT-3, SCUT-O*cdo-*O*sep39* on 48h.

### 4.1 Determination of keratinase activities

Fig. 7 showes the keratinase activities of SCUT-3 and this 4 recombinant groups in in 10% CFM fermentation at different times. From the picture, we can see SCUT-O*cdo*-p24880-O*sep39* demonstrated highest keratinase activity at 48 h. This affirmed that *p24880* can be further used to replace *ermE* for feather degradation.

![](https://static.igem.org/mediawiki/2021/d/d2/T--GDSYZX--Results-fig12.png)

Figure 7 Keratinase activities detected in SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at different time.

### 4.2 Determination of amino acid content

According to Fig. 8, at the first 24h, the SCUT-O*cdo*-p24880-O*sep39* has already exhibited high amino acid content. At 48 h, the SCUT-O*cdo*-p24880-O*sep39* still showed highest amino acid content among all five groups, which is over 35000 μg/mL, and significantly higher than SCUT-3 and SCUT-O*cdo-*O*sep39*.

![](https://static.igem.org/mediawiki/2021/1/17/T--GDSYZX--Results-fig7.png)

Figure 8 Amino acid content from SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at 24 h and 48 h, \*P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

### 4.3 Determination of peptide content

Figure 9 shows the content of peptide for this 5 groups in 10% CFM fermentation at 24 h and 48 h. SCUT-O*cdo*-p24880-O*sep39* exhibited highest content both at 24 h and 48 h, which is approximately 22 mg/mL at 48 h. The peptide content of SCUT-O*cdo*-p24880-O*sep39* is significantly higher than SCUT-3 and SCUT-O*cdo-*O*sep39*.

![](https://static.igem.org/mediawiki/2021/1/15/T--GDSYZX--Results-fig8.png)

Figure 9 Peptide content from SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at 24 h and 48 h, \*P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

### 4.4 Determination of peptide and amino acid content

Figure 10 shows the feather protein conversion for this 5 groups in 10% CFM fermentation at 24 h and 48 h. SCUT-O*cdo*-p24880-O*sep39* exhibited highest content both at 24 h and 48 h, which is approximately 57000 μg/mL at 48 h. The peptide and amino acid content of SCUT-O*cdo*-p24880-O*sep39* is significantly higher than SCUT-3 and SCUT-O*cdo-*O*sep39*.

![](https://static.igem.org/mediawiki/2021/c/c6/T--GDSYZX--Results-fig9.png)

Figure 10 Peptide and amino acid content from SCUT-3 and recombinant SCUT-3 in 10% CFM fermentation at 24 h and 48 h, \*P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

### 4.5 Analysis of feather degradation rate

Figure 11 shows the degradation rate for different groups. SCUT-O*cdo*-p24880-O*sep39* showed highest degradation rate; SCUT-O*cdo*-p24880-O*sep39* had the highest feather-degrading ability. The degradation rate of SCUT-O*cdo*-p24880-O*sep39* was significantly higher than SCUT-3, but has no significant difference with SCUT-O*cdo*-p1380-O*sep39*.

![](https://static.igem.org/mediawiki/2021/3/36/T--GDSYZX--Results-fig10.png)

Figure 11 Comparison of the degradation rate between SCUT-3 and recombinant SCUT-3; \*P < 0.05. P-values between groups were obtained by unpaired two-tailed Student’s t-test. All data were presented as mean ± SD.

## 5. Growth conditions of various SCUT-3 strains

Earlier studies found that co-overexpression of *cdo1* and *sep39* under constitutive promoter ermE resulted in SCUT-O*cdo-*O*sep39* yielding fewer cells than wild type SCUT and SCUT-O*sep39* but more than SCUT-O*cdo*. In our experiments, the bacterial growth condition tests showed that the overexpression of *sep39* under promoter *p24880* did not affect wild type SCUT’s growth (no significant difference compared to the wild-type) which demonstrated that the negative effect of *cdo1* overexpression disappeared.

![](https://static.igem.org/mediawiki/2021/d/d9/T--GDSYZX--Results-fig11.png)

Figure 12 Bacterial growth condition of wild type SCUT-3 (A), SCUT-O*cdo-*O*sep39* (B), SCUT-O*cdo*-p1380-O*sep39* (C), SCUT-p22610-O*cdo-*O*sep39* (D) and SCUT-O*cdo*-p24880-O*sep39* (E).

## Future Prospects and Concern

Because of the resulting pollution, amino acid destruction, low solubility and difficult to be digested by animals, traditional physical/chemical treatment methods for feather waste are gradually being abandoned[1]. The most promising alternative approaches involve biodegradation using bacteria. Through studies of using microbial means to convert accumulated millions tonnes of feathers into keratin, we take one step further to accomplish turning feather, instead of a burdensome waste, into rich, widely usable resource of protein and valuable raw materials for many fields.

(1) Biomedical applications of keratin. Keratin based biomaterials have been widely produced and used in various biomedical applications, such as bone tissue engineering[2, 3], wound healing[4, 5], nerve regeneration[6], skin replacement[7] and controlled drug delivery[8-10]. Our research is a simple, green, cost effective and yet efficient methodologies for the better extraction of keratin from feather. It is expected that keratin we extracted will turn into a mainstream biomaterial for clinical trials.

(2) Feather is a natural source of amino acids, peptides, and minerals that can be used in animal feed supplements and as a nitrogen fertilizer for plants[11]. Biodegradation of feather by microbial is a safe, sanitary and cost-effective technology that can play an important role in in processing feather waste.

Our study would shed light on the feather utilization mechanisms of FDB and provide a highly efficient, cost-effective way for degradation of feathers and extraction of available keratin, amino acids. Further exploration of the physiological functions of CDO and Sep39 in this Streptomyces. SCUT-3 will likely provide additional insights into biodegradation using bacteria.

## Reference

[1] Li Z W, Liang S, Ke Y, et al. The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3 [J]. Commun Biol, 2021, 3(1): 191.

[2] Li J S, Li Y, Liu X, et al. Strategy to introduce an hydroxyapatite-keratin nanocomposite into a fibrous membrane for bone tissue engineering [J]. J Mater Chem B, 2013, 1(4): 432-7.

[3] Tachibana A, Nishikawa Y, Nishino M, et al. Modified keratin sponge: Binding of bone morphogenetic protein-2 and osteoblast differentiation [J]. Journal of Bioscience and Bioengineering, 2006, 102(5): 425-9.

[4] Ham T R, Lee R T, Han S, et al. Tunable keratin hydrogels for controlled erosion and growth factor delivery [J]. Biomacromolecules, 2016, 17(1): 225-36.

[5] Nakata R, Tachibana A, Tanabe T. Preparation of keratin hydrogel/hydroxyapatite composite and its evaluation as a controlled drug release carrier [J]. Mater Sci Eng C Mater Biol Appl, 2014, 41(59-64.

[6] Lin Y C, Ramadan M, Van Dyke M, et al. Keratin gel filler for peripheral nerve repair in a rodent sciatic nerve injury model [J]. Plast Reconstr Surg, 2012, 129(1): 67-78.

[7] Xu S, Sang L, Zhang Y, et al. Biological evaluation of human hair keratin scaffolds for skin wound repair and regeneration [J]. Mater Sci Eng C Mater Biol Appl, 2013, 33(2): 648-55.

[8] Yin X C, Li F Y, He Y F, et al. Study on effective extraction of chicken feather keratins and their films for controlling drug release [J]. Biomater Sci, 2013, 1(5): 528-36.

[9] Guo J, Pan S, Yin X, et al. pH-sensitive keratin-based polymer hydrogel and its controllable drug-release behavior [J]. Journal of Applied Polymer Science, 2014.

[10] Shavandi A, SilVA T H, Bekhit A A, et al. Keratin: dissolution, extraction and biomedical application [J]. Biomater Sci, 2017, 5(9): 1699-735.

[11] Kornillowicz-kowalska T, Bohacz J. Biodegradation of keratin waste: Theory and practical aspects [J]. Waste Manag, 2011, 31(8): 1689-701.

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