EXPERIMENT-BUILD

Micro-Organisms Kind

We tried to modify a type of micro-organism that is able to degrade feathers efficiently. The name of this organism is Streptomyces sp. SCUT-3.

Promoters and Plasmids

In order to select the well-characterized promoters, we screened the promoters based on RNA-Seq data, using TPM value as gene expression intensity evaluation index. Those with higher TPM value are considered as promoters’ candidate and were amplified by PCR later.Under this condition, we selected pro1380, pro2953, pro3035, pro3040, pro3071, pro15290, pro22610, pro 24880 as our candidate promoters.

Table 1. Names of promoters and base pair

Name of Promoters	Base Pair(bp)
pro1380	300
pro2953	303
pro3035	403
pro3040	263
pro3071	193
pro15290	218
pro22610	152
pro24880	461

The PCR reaction systems are as follows:

Reagent	Volume (μL)
Master Mix	45
genome DNA	1
Primer F	2
Primer R	2

The procedure of PCR reaction began are showing as follows:

Agarose gel electrophoresis

1% agarose gel electrophoresis was used for detection the target band. After adding 0.25 g of agarose into a 250 mL Erlenmeyer flask, we poured 25 mL of 1×TAE solution into this Erlenmeyer flask. Later, we put the Erlenmeyer flask into a microwave oven and heated for 30-60 s until the agarose is completely dissolved. After the solution is cooled, we added 0.25 μL nucleic acid dye into this solution. We then poured this solution into the glue tank where the comb and the bottom plate have been inserted, and let it stand for about 30 min. After the agarose gel has solidified, we placed it in an electrophoresis tank containing 1×TAE to ensure the gel is completely immersed in the buffer. Then we added the DNA sample into the gel well and electrophoresis at 120 V for about 20 min.

Enzyme digestion experiment

The restriction expression vector in our experiment contains 5 μL of 10×H buffer, 1 μg of pSET152, 3 μL of NdeI, 3 μL of NotI, and sterile water.

Reagent	Volume
10 ×H buffer	5 μL
pSET152	1 μg
NdeI	3 μL
NotI	3 μL
water	Up to 50 μL

Vector - target gene recombination experiment

Target gene fragment and linearized vector recombination experiments were done by adding 5 μL of 2×SoSoo Mix (Trelief™ SoSoo Cloning Kit Ver.2), 0.02 pmol of linearized plasmid, 0.1 pmol of the target gene of pSET152, and water. The mixture was incubated at 50℃ for 30 min.

Reagent	Volume
2 × SoSoo Mix	5 μL
Linearized plasmidp	0.02 pmol
target gene of SET152	0.02 pmolL
water	Up to 10 μL

Plasmid transformed Escherichia coli DH5α

(1) Mixed 50 μL E.coli DH5α and 5 μL vector - target gene recombination produst gently and held on ice for 30min.

(2) In 42℃ water bath heat shock bacteria 90 s, and qucinkly put the mix into the wet ice. Let it stand for 2 min.

(3) Added 1 mL liquid LB medium into the mix and incubate it at 37℃ constant temperature box table, 220 rpm shaking the culture for 1 h.

(4) Centrifuged the culture at 5000 rpm for 2 min and discard the supernatant.

(5) Resuspended the bacterial residue with 100 μL sterile LB and spread on LB plate containing apramycin, then incubate overnight at 37℃ for 10-12 h.

(6) Selected the positive clones by using colony PCR.

**Conjugative transfer between Streptomyces and E.coli ET12567/pUZ8002 Donor bacteria preparation**

E.coli ET12567/pUZ8002 with integrated plasmid pSET152 was inoculated in 10 mL of LB liquid medium (50 μg/mL kanamycin, 25 μg/mL chloramphenicol, 50 μg/mL apramycin) at 37℃, 220 rpm for 10-12 h. Later, transfering 5mL above cultured bacteria liquid to a 100 mL LB liquid medium and incubate at 37℃ until the OD600 reaches 0.4. Centrifuged at 5000 rpm for 5 min to collect the bacteria, and washed the bacteria twice with 50 mL of non-anti-LB. After that resuspended them in 10 mL of anti-LB for use.

Receptor bacteria preparation

Obtaining spores: Used a toothpick to gently take the spores on the solid medium that has been cultured for more than one week, and place them in water. Add sterilized glass beads and shaked with a shaker until the spores were evenly suspended in the water. If cryopreserved spores needed to be heat shocked first: mix 0.5 mL of spore suspension with 2 mL of TES, and heat shock at 50℃ for 10 min. After cooling to room temperature, added 2.5 mL of 2×spore pre-germination medium and pre-germinate at 37℃, 200 rpm for 3 h. The spores of the recipient strain SCUT-3 were controlled at 107 cfu/mL.

Mixed the donor and recipient bacteria at a ratio of 10:1, centrifuge at 5000 rpm for 2 minutes and spread them on MS plates containing different magnesium ion concentrations. After incubation at 37℃ for 12–16 h, the plates were covered with 1 mL water containing 1 mg nalidixic acid and 1 mg apramycin. The cells were incubated at 37℃ for 3–5 d until the exconjugants appeared. The clones were verified by colony PCR.

Method of the SCUT-3 fermentation (10% CFM)

Transferring the seed solution into 10% CFM and leaved them to ferment at 40℃ for 6 d. After fermentation, added 200 mL of distilled water into it. Taken 2 mL of fermentation broth and centrifuged at 10000 rpm for 5 min at 4℃. The supernant were further used for determination of keratinase activity, sulfhydryl content, amino acid peptide content.

Team:GDSYZX/Build