Team:GDSYZX/Engineering
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(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(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(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(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(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(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/ResultsSTEP 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-Ocdo-Osep39 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-Ocdo-Osep39. ③Testing the bacterial growth condition.
https://2021.igem.org/Team:GDSYZX/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-Ocdo-Osep39.
https://2021.igem.org/Team:GDSYZX/Test(4) Learn
The feather degradation efficiency of recombinant strain SCUT-Ocdo-p24880-Osep39 was higher than wild-type SCUT-3 and SCUT-Ocdo-Osep39 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/ResultsConclusion
We successfully that the SCUT-Ocdo-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-Ocdo-p24880-Osep39 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/ResultsFurther 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.