iGEM 2022 — Team gxu-china

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Notebook

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### notebook-1.pdf

[Download](https://static.igem.wiki/teams/4354/wiki/notebook/notebook-1.pdf)

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### notebook-2.pdf

[Download](https://static.igem.wiki/teams/4354/wiki/notebook/notebook-2.pdf)

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Engineering

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Experiments

### Materials and source

[1) Bacteria
strains](#accordion1-card-body-junpxiod)

1)

Bacillus subtilis

168 (Bs/WT): obtained from Dr. Wei Zeng lab.

2) DH5α, obtained from Dr. Weihui Li lab.

3) Bs/pHY-P43

-nar

, constructed by this experiment.

4) Bs/pHY-P43-P

BLind-v1-amo-hao-nirS,

constructed by this experiment.

5) Bs/pHY-P43-

nar/

pUC19-P

cecG2-veg-nucA,

constructed by this experiment.

6) Bs/pHY-P43-P

BLind-v1-amo-hao-nirS/

pUC19-P43

-el222-

P

cecG2-veg-nucA,

constructed by this experiment.

[2) Genes and
promoters](#accordion1-card-body-jwureenv)

1)

el222,

sequence obtained from National Center for Biotechnology Information (NCBI).

2)

nar,

sequence obtained from NCBI.

3)

rbs34, amo, hao, nirS,

sequence obtained from NCBI (with

P

BLind-v1).

4)

nucA,

sequence obtained from

sequence obtained from NCBI.

5)

gfp,

amplified from pHY300PLK-P

veg-gfp

which obtained from ZJU-China.

6)

P43,

sequence obtained from NCBI.

7)

P

BLind-v1,

sequence obtained from NCBI.

8)

P

cecG2-veg,

sequence obtained from NCBI.

[3) Plasmids](#accordion1-card-body-syapgqiv)

![](https://static.igem.wiki/teams/4354/wiki/experiments/plasmids.png)

[4) Primers](#accordion1-card-body-uwxlrkro)

All primers were purchased from Tsingke Biotechnology Company.

![](https://static.igem.wiki/teams/4354/wiki/experiments/ordinary-primer.png)

![](https://static.igem.wiki/teams/4354/wiki/experiments/q-pcr-primer.png)

[5) Enzymes and antibiotics](#accordion1-card-body-uwxlrkr1)

1)

Xma

I, purchased from NEB Company (USA).

2)

BamH

I, purchased from TAKARA Company (CHN).

3)

Hind

III, purchased from TAKARA Company (CHN).

4)

Xba

I, purchased from TAKARA Company (CHN).

5)

Pci

I, purchased from NEB Company (USA).

6)

EcoR

I, purchased from TAKARA Company (CHN).

7) Ampicilin (Amp), purchased from solarbio Company (CHN).

8) Tetracycline (Tet), purchased from solarbio Company (CHN).

[6) Reagent](#accordion1-card-body-uwxlrkr2)

1)Tris-base, purchased from sigma Company (GER).

2) Glycerinum, purchased from sigma Company (GER).

3) NaOH, purchased from Macklin Company (CHN).

4) CaCl

2,

purchased from APS Company (USA).

5) MgCl

2,

purchased from APS Company (USA).

6) Tryptone, purchased from aladdin Company (CHN).

7) Yeast extract, purchased from aladdin Company (CHN).

8) NaCl, purchased from aladdin Company (CHN).

9) Agar powder, purchased from solarbio Company (CHN).

10) Extraction kit, purchased from Omega Company (USA).

11) Agarose: purchased from Sangon Biotech Company.

12) EDTA, purchased from Sangon Biotech Company (CHN).

13) SDS, purchased from Sangon Biotech Company (CHN).

14) potassium acetate, purchased from Sangon Biotech Company (CHN).

15) Glacial acetic acid, purchased from Sangon Biotech Company (CHN).

16) Absolute ethyl alcohol, purchased from Sangon Biotech Company (CHN).

17) 75% ethanol, purchased from Sangon Biotech Company (CHN).

18) Twain, purchased from Sangon Biotech Company (CHN).

19) Glucose, purchased from Sangon Biotech Company (CHN).

20) EGTA, purchased from Sangon Biotech Company (CHN).

21) (NH

4

)

2

SO

4

: attained from Dr. Wei Zeng lab.

23) K

2

HPO

4

: attained from Dr. Wei Zeng lab.

23) KH

2

SO

4,

attained from Dr. Wei Zeng lab.

24) Casein amino acid, attained from Dr. Wei Zeng lab.

25) Trisodium citrate, attained from Dr. Weihui Li lab.

26) MgSO

4

·7H

2O,

attained from Dr. Wei Zeng lab.

27) EASY spin Plus, purchased from Aidlab Company (CHN).

28) PBS, purchased from Aladdin Company (CHN).

29) 6×Loading Buffer, purchased from TAKARA Company (CHN).

30) Marker, purchased from TAKARA Company (CHN).

### Protocol

[1. Activation of glycerol bacteria stock and Cultivation of single colonies](#accordion1-card-body-junpxio4)

1) Activation of glycerol bacteria stock, 5 mL of liquid medium, 5 μL of corresponding antibiotic and 100 μL of glycerol bacteria were added to sterile 30 mL cylindrical glass bottles, and incubated overnight in shaker at 37 ℃, 160 rpm/min.

2) Cultivation of single colonies, picked out the single colonies on the plate by a 10 μL tip, and then the tip was put into a 30 mL cylindrical glass bottle with 5 mL liquid medium and 5 μL of corresponding antibiotics. Finally, the 30 mL cylindrical glass bottle was incubated overnight in shaker at 37 ℃, 160 rpm/min.

[2. Preparation of chemically competent cells of

Escherichia coli (E.coli)](#accordion1-card-body-jwureen1)

1) A tube of 100 μL DH5α was inoculated in 5 mL LB liquid medium and incubated overnight in shaker at 37 ℃, 160 rpm/min.

2) Inoculated the overnight culture in 100 mL of LB medium at 1% ratio and incubated at 37 ℃ for 2-3 h (Cultured to OD

600

=0.5-0.6).

3) Cooled the bacteria culture in the mixture of ice and water for 5-10 minutes in advance. Then transferred the treated bacteria culture to a centrifugal tube and placed it on ice for 10 mins to cool the bacteria liquid.

4) Transferred the cold bacterial liquid into 4 ℃ centrifuged at a speed of 6000 rpm/min for 5 mins. Discarded the liquid supernatant and collected the bacterium.

5) Added 60 mL cold 0.1 mol/L CaCl

2

solution to re-suspend the bacterium, and then cooled it in an ice bath for 45 mins.

6) Transferred the bacterial liquid into a 4 ℃ centrifuged at a speed of 6000 rpm/min for 5 mins to discard the liquid supernatant and collected the bacterium.

7) Added 1 to 2 mL cold 15% glycerol solution (containing 0.1 mol/L CaCl

2

solution) to re-suspend the bacterium, and divided them into 300 μL of each micro centrifuge tube and then stored at -80 ℃.

[3.Transformation of DH5α chemically competent cells](#accordion1-card-body-syapgqi1)

1) Added an appropriate amount of DNA sample or ligated reaction product to 100 μL competent cells, mixed slightly, and took an ice bath for 30 mins.

2) The micro centrifuge system was placed in a water bath at 42 ℃ for 90 s incubation.

3) After incubation, placed the competent cells quickly in the mixture of ice and water for cooling 2 mins, and then added into 800 μL LB liquid medium for shaking culture at 37 ℃ for 45-60 mins.

4) Took out, centrifuged at a speed of 8000 rpm/min for 1 min, removed the liquid supernatant, and left 100 μL.

5) Coated on LB solid plate containing corresponding antibiotics and cultured upside down overnight in 37 ℃ incubator to observe the growth of transformation.

[4. Plasmid extraction of

E.coli](#accordion1-card-body-uwxlrkr1)

1) Inoculated

E.coli

strains containing the plasmid in 5 mL of LB liquid medium with selective antibiotics and then cultured in a shaking table at 37 ℃, 160 rpm for 12-16 h.

2) Transferred the bacterial solution to the micro centrifuge tube, centrifuged it at a speed of 8000 rpm/min at 4 ℃ for 1 min, discarded the liquid supernatant, and under the same conditions until all the bacterial solutions were transferred.

3) Added 100 μL alkali lysate I to the bacteria and suspended fully.

4) Added 200 μL alkali lysate II, slowly inverted mixed, and flicked the bottom, until the solution became clear, then added 150 μL alkali lysate III, reversed and mixed micro centrifuge system, stood at 4 ℃ for 5 mins, then 4 ℃ centrifuged at a speed of 12000 rpm/min for 5 mins.

5) Transferred the liquid supernatant to a clean centrifugal tube, added anhydrous ethanol of 2 times volume of the liquid supernatant, mixed and precipitated at 4 ℃ for 5 mins, then centrifuged at a speed of 12000 rpm/min for 5 mins, and discarded the liquid supernatant.

6) Added 500 μL 75% ethanol solution to the precipitation, discarded the liquid supernatant after 4 ℃ centrifuged at a speed of 12000 rpm/min for 2 mins, and placed at room temperature to volatilize the remaining liquid.

7) Added an appropriate amount of sterile water at about 60 ℃, and the shaker oscillated evenly, and stored the plasmid solution at -20 ℃.

[5. Preparation of chemicals](#accordion1-card-body-uwxlrkr6)

1) Alkali lysate I, weighed 3.03 g Tris-base, 2.92 g EDTA, 9.9 g glucose into 1 L H

2O, adjusted pH to 8.0 , 121 ℃ sterilized for 10 mins then stored at 4 ℃.

2) Alkali lysate II, mixed 0.4 mol/L NaOH and 2% SDS in equal volume, while 0.4 mol/L NaOH and 2% were prepared by sterile water respectively and stored at room temperature.

3) Alkali lysate III, weighed 194.42 g potassium acetate into 800 mL steriled water, then 115 mL of acetic acid was added, and finally the volume was fixed to 1 L, stored at 4 ℃.

4) 50% glycerol, equal volume of 100% glycerol and sterile water were mixed and stored at 4 ℃.

5) 2×GC buffer: added BSA 40 mg, Betaine 24.6 g, Tris-HCl 4 mL at pH 8.8, 1 mol/L KCl 4 mL, 800 μL of 1 mol/L Mg

2

SO

4

at pH 8.8, 12 mL of 0.5 mol/L (NH

4 )2SO4

at pH 8.8, 24 mL 50% glycerin, 4 mL DMSO, 4 mL N, N, N-dimethylformamide, Triton X-100 400 μL, and finally the volume was fixed to 200 mL with sterilized water.

6) SPI salts, weighed (NH

4 )2

SO

4

0.2 g, K

2

HPO

4

1.4 g, KH

2

PO

4

0.6 g, Na

3

C

6

H

5

O

7

·2 H

2O, Mg

2SO 4·7 H2O 0.02 g and finally the volume was fixed to 100 mL with ddH2O, sterilized at 121 ℃ for 20 mins, and stored at room temperature.

7) Casein amino acid/yeast extract, weighed Casein amino acid 0.2 g, yeast extract 1 g , then the volume was fixed to 10 mL with ddH

2O, then filtered and sterilized, and stored at room temperature after completion.

8) 50 mM CaCl

2, weighed CaCl2

0.111 g and dissolved it in 20 mL ddH

2O, and sterilized it at 121 ℃ for 20 mins, then stored at room temperature.

9) 250 mM MgCl

2,

weighed MgCl

2·6H2O 0.5075 g and dissolved it in 20 mL ddH2O, and sterilized it at 121 ℃ for 20 mins, then stored at room temperature.

10) 100 mM EGTA, weighed ethyleneglycol-bis-(2-aminoethylether)-N,N'-tetraacisicacid (EGTA) 0.38 g and dissolved it in 10 mL ddH

2

10)O, and adjusted pH to 7 with NaOH then filtered and sterilized, finally stored at room temperature after completion.

[6. DNA/RNA extraction using commercial kits](#accordion1-card-body-uwxlrkr7)

All reagent below referent was offered by Extract kit.

1) Added twice the volume of Binding Buffer to the sample, stood at room temperature for 1 min. Transferred to adsorption column, then centrifuged at a speed of 13000 rcf/min for 1 min and discarded the liquid supernatant of collect column (If the sample was attained from the gel extraction, the volume of Binding Buffer was exceeded the gel).

2) Added 300 μL Binding Buffer to adsorption column, then centrifuged at a speed of 10000 rcf/min for 1min and discarded the liquid supernatant of collect column.

3) Added 700 μL Washing Buffer to adsorption column, stood at room temperature for 2 mins, then centrifuged at a speed of 10000 rcf/min for 1 min and discarded the liquid supernatant of collect column (Repeated this step twice).

4) Added nothing to adsorption column, stood at room temperature for about 5 mins, then discarded collect column and transferred adsorption column to a new centrifuge tube.

5) Added 30~50 μL 55℃ ddH

2O, stood at room temperature for 1 min and then centrifuged at a speed of 13000 rcf/min for 1 min.

6) Transferred the water in the centrifuge tube to adsorption column and then centrifuged one more time.

7) Discarded the adsorption column, and restored the sample at -20 ℃.

[7. Gel extraction](#accordion1-card-body-uwxlrkr8)

1) Sample Preparation, added electrophoretic Loading buffer, Gel Loading Dye, Purple(6×) into the products of endonuclease digestion, that was, added 1part buffer to 5 parts of sample.

3) Electrophoresis, completely immersed the agarose gel in the buffer, added mark and the sample into the loading well, and performed electrophoresis at 100 V for 45 mins.

4) Stain, placed the gel in 10 μg/mL EB dye solution for 2-4 mins at room temperature.

5) Observation and cutting, observed the electrophoresis tape and its position on the ultraviolet fluoroscope, and cut the fragments in the required range.

[8. DNA gel electrophoresis](#accordion1-card-body-uwxlr1r8)

1)Sample Preparation, added electrophoresis Loading buffer, Gel Loading Dye, Purple (6×) to nucleic acid sample, that was, added 1 part buffer to 5 parts of sample.

2) Agarose gel Preparation, weighed 0.8 g agarose, added 100 mL of TAE(1×) buffer，prepared 0.8% agarose, heated it in a microwave oven for several minutes to completely melted the agarose, mixed it well, poured it into a gel making mold, inserted a comb with a suitable aperture, and after the gel solidified, pulled out the comb and put it into an electrophoretic tank containing TAE(1×) buffer.

3) Electrophoresis, completely immersed the agarose gel in the buffer, added mark and the sample into the loading well, and performed electrophoresis at 120 V for 30 mins.

4) Imaging, dyed the electrophoretic gel in EB for 2 mins, then performed UV imaging in the GelDoc Go Imaging System, and took photos for recording.

[9. Preparation of LB medium](#accordion1-card-body-uwx2r1r8)

1) Weighed NaCl 10 g, Tryptone 10 g, Yeast extract 5 g then the volume was fixed to 1 L with ddH

2O and adjusted pH to 7.0 with NaOH, sterilized at 121 ℃ for 20 mins, and stored at room temperature.

2) For solid LB medium, 1.5 g Agar powder was added every 100 mL on the basis of (1).

[10. Preparation of medium containing antibiotics](#accordion1-card-body-uwx2r1r1)

1) For the liquid medium, the corresponding antibiotics were added at a ratio of 1/1000 before using.

2) For the solid medium, it was heated and melted before using. When the temperature was reduced to about 50-60 ℃, the corresponding antibiotics were added in the proportion of 1/1000, which can be used to make the plate.

[11. Preparation of chemically competent cells of

Bacillus subtilis](#accordion1-card-body-uwx2r1r1)

1) Took out a tube of chemocompetent cells, melted them on ice and added about 1 μg of DNA.

2) Incubated in shaker at 37 ℃, 160 rpm for 90 mins.

3) Added 300 μL LB medium and then incubated in shaker at 37 ℃, 160 rpm for 40 mins.

4) Took 50 to 100 μL to coat on LB solid plate containing antibiotics and cultured upside down overnight in 37 ℃ incubator to observe the growth of transformation.

[12. PCR](#accordion1-card-body-uwx2r1r1)

1) Preparation 1-reaction PCR mix into the PCR tube by adding together 8.5 µL deionized water, 12.5 µL 2×GC buffer, 2 µL dNTP mix,1 μL templated DNA, 1 μL primer pairs and 0.125 µL rTaq DNA polymerase.

2) Placed the PCR tubes into the PCR apparatus and run it (Protocol can be divided into five centrifuge tubes, pre-denaturation → denaturation → anneal→ extension → final extension). After the PCR program entered denaturation, it run 30 times successively through denaturation, annealed and extension, and finally entered complete extension. After completing extension, waited for the temperature to drop to 16 ℃ to end the pro-gram.

① Pre-denaturation, run at 95 ℃ for 5-10 mins to completely denature the primes, so as to ensure that the primers were single stranded. Meanwhile, some secondary structures of the template DNA aredestroyed, and the double stranded of the template DNA was fully dissociated.

③ Anneal, reduced the solution temperature to the appropriate temperature (determined by annealing temperature pf the primer) and centrifuged it for 20 s to make the template DNA and primer complemented each other according to the principle of base pairing principle.

④ Extension, when the reaction temperature of the solution rose to 72 ℃, the rTaq DNA polymerase took the single strand DNA as the template and centrifuged the complementary DNA in the direction of 5'- 3' by using four kinds of deoxynucleoside triphosphate (dNTP) in the reaction mixture under the guidance of primers. The extension time dmicrocentrigugeended on the length of the fragment. The PCR instrument used in this experiment was extended by 1 kb/min.

⑤ Final extension, maintained it at 72 ℃ for 8 mins to make PCR reaction more completed to improve amplification yield.

⑥ Waited for the temperature to drop to 16 ℃ and the PCR procedure was completed.

[13. QuantitativeReal-time PCR](#accordion1-card-body-uwx2r1r4)

1)All reagent below referent was offered by EASY spin Plus.

2)Extract RNA, ① Centrifuged the strains at a speed of 8000 rpm/min for 2 mins, and discarded the liquid supernatant. ② Added moderate lysozyme(about 100~200 μL) and cultured in 37 ℃ incubator for 30 mins. During the period, turned upside down and mixed for every five minutes. ③ Added 500 μL RLT Plus lysate and mixed sufficiently, shook violently for 20 s. ④ Added the lysate to DNA clear column, then centrifuged at 1300 rpm for 30 s, and retained the filtrates. ⑤ Added 500 μL 70% ethanol solution to the filtrates. ⑥ Added the mixture to the absorption column and then centrifuged at 13000 rpm for 30 s. ⑦ Added 700 μL free protein serum, stood at room temperature for 30s and then centrifuged at 13000 rpm for 30 s. ⑧ Added 500 μL rinse and centrifuged at 13000 rpm for 30 s twice. ⑨ Centrifuged without anything at 13000 rpm for 2 mins. ⑩ Transfer absorption column to centrifuge tube which has been heated at 121 ℃ for one hour. And then added 30~50 μL RNase-free H

2O, stood at room temperature for 1 min and centrifuged at 13000 rpm for 1 min. Took out 5 μL for electrophoresis and measuring the concentration quickly, 8 μL for reverse cDNA and the others for storage.

3) Prepare the sample with DNaseⅠsystem (10 μL), 80%RNA, 10% DNaseⅠ, 10% 10× Buffer. Stood system at 37 ℃ for 30mins and 65 ℃ for 10 mins, then terminated reaction and stored on ice.

4) Reverse cDNA, Different RNA are adjusted by volume and concentration to the same amountof material. System, RNA, 500 ng, Random Primer, 1 μL, 5× TRUE Reaction Mix, 4 μL, added RNase-free H

2 O to 20 μL. Every type RNA prepared 4 PCR tubes, each PCR tube contained 40 μL. Stood the system at 42 ℃ for 15 mins and at 85 ℃ for 5 s.

5) q-PCR, ① A tube of 100 μL transformant was inoculated in 5 mL LB liquid medium and incubated in shaker at 37 ℃ , 160 rpm/min (Cultured to OD

600=1.0). ② Inoculated the culture in 100 mL of LB medium at 1% ratio and incubated at 37 ℃ for 2-3 h (Cultured to OD

600=1.0). ③ Transferred the bacterial liquid into 4 ℃ centrifuged at a speed of 6000 rpm/min for 5 mins. Discarded the liquid supernatant and collected the bacterium. ④ Washed the bacterium with PBS twice, and then suspended fully with moderate PBS. ⑤ Added the sample to microplate reader, adjusted parameter and attained the data.

5) q-PCR, ① A tube of 100 μL transformant was inoculated in 5 mL LB liquid medium and incubated in shaker at 37 ℃ , 160 rpm/min (Cultured to OD

2O 9.5 μL.

[14. Enzyme digestion](#accordion1-card-body-uw12r1r4)

1) Single Enzyme digestion system of plasmid, 80% plasmid, 10% buffer, 5% enzyme, 5% ddH

2O.

2) Double Enzyme digestion system of plasmid, 80% plasmid, 10% buffer, 5% enzyme A, 5% enzyme B.

3) Single enzyme digestion system of gene fragment, 80% plasmid, 10% buffer, 2% enzyme, 8% ddH

2O.

4) Double enzyme digestion system of gene fragment, 80% gene fragment, 16% buffer, 2% enzyme A, 2% enzyme B.

[15. Enzyme-linked Ligation](#accordion1-card-body-uwx2r1r5)

1) Enzyme-linked system 1 (total volume 10 μL), 20% plasmid, 60% gene fragment, 10% buffer, 10% T4 DNA ligase.

2) Enzyme-linked system 2 (total volume 10 μL), 40% plasmid, 40% gene fragment, 10% buffer, 10% T4 DNA ligase.

3) Reaction condition, stood at 16 ℃ for 4 hours or overnight.

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Results

Order of constructing plasmids

![]( https://static.igem.wiki/teams/4354/wiki/results/result1.png )

Strains constructed with

Bacillus subtilis

168 (Bs/WT)

Bs/pUC19-P43

-el222-

P

cpcG2-veg-nucA/

pHY-P43-P

BLind-v1-amo-hao-nirS

Bs/pUC19-P

cpcG2-veg-nucA/

pHY-P43-

nar

![](https://static.igem.wiki/teams/4354/wiki/guide.png)

* [1. Construction of expression system of candidate genes involved in nitrogen removal](#tabs-1-1)
* [2. Construction the switch of blue light metabolic pathway](#tabs-1-2)
* [3. Detection of candidate genes expression](#tabs-1-3)
* [4. Construction of candidate genes expression vector based on green light regulation](#tabs-1-4)
* [5. Construction the switch of green light metabolic pathway](#tabs-1-5)

### 1.Construction of target gene expression vector based on blue light Regulation

We selected four catalytic enzymes coding genes

amo

(for coding AMO),

hao

(for coding HAO),

nar

(for coding NAR),

nirS

(for coding NIR) as candidate genes which play important roles in the denitrogenation pathway. The four catalytic enzymes catalyze ammonium salt, hydroxylamine, nitrate and nitrite to nitric oxide, respectively

(Fig. 1.1)

. In order to control the gene expression under blue light, we applied P

BLind-v1

promoter that is driven by a blue light responsive protein EL222. The blue light triggers the conformational change of EL222, making it bind the P

BLind-v1,

which induces the candidate gene expression. In contrast, when in the dark (off state), EL222 cannot bind the P

BLind-v1

and the candidate gene will be silenced. The detailed design can be referred to

Fig. 1.2

.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-1.png)

Fig. 1.1

Metabolic pathway of nitrogen removal by heterotrophic nitrification and aerobic denitrification. AMO: Ammonia monooxygenase, HAO: Hydroxylamine oxidase, NXR: Nitrite oxidoreductase, NAR: Nitrate reductase, NIR: Nitrite reductase, NOR: Nitric oxide reductase, NOS: Nitrous oxide reductase.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-2.png)

Fig. 1.2

Design and characterization of the blue light inducible gene expression system. Mechanism diagram of blue light inducible system, the blue light inducible promoter (P

BLind-v1

), consists of EL222 binding region fused to the

lux

I promoter.

#### Construction of nar expression system

Design

Cloned

nar

into the expression vector pHY-P43 containing a constitutive promoter P43 commonly used in Bs/WT, shown in

Fig. 1.3.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-3.png)

Fig. 1.3

The construction of P43

-nar.

Build

Obtained and verified the

nar

as shown in

Fig. 1.4A.

According to the restriction endonuclease sites, used the corresponding restriction enzymes to digest nar and pHY-P43, where the digested pHY-P43 vector was shown in

Fig. 1.4B.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-4.png)

Fig. 1.4 (A)

Lane 1: DNA Marker, Lane 2:

nar

gene fragment, amplified by rTaq DNA polymerase at annealing temperature 61 ℃, and the band size was as expected.

(B)

Lane 1: DNA Marker, Lane 2: pHY-P43 vector digested with

BamH

Ⅰ and

Xma

Ⅰ, and the band size was as expected.

Test

The colonies were selected from the transformation plate and inoculated into the medium containing Tetracycline for overnight culture. Extracted the plasmid and carried out verification of the size of pHY-P43

-nar

and verification of the

nar.

The plasmid verification results were shown in

Fig. 1.5.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-5.png)

Fig. 1.5

Validation of pHY-P43

-nar.

(A) Comparison of plasmid size between pHY-P43 (4.3 kb) and pHY-P43

-nar

(6.6 kb), Lane1: pHY-P43, Lane2: pHY-P43

-nar.

(B) Validation of

nar

used pHY-P43

-nar

as the template, Lane 1: DNA Marker, Lane 2:

nar.

Learn

Fig. 1.5

showed that we had successfully constructed pHY-P43

-nar

which provided possibility for further improvement of nitrogen removal efficiency.

#### Construction of gene cluster (including amo, hao and nirS ) expression system

Design

Cloned gene cluster into pHY-P43, as shown in

Fig. 1.6.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-6.png)

Fig. 1.6

The construction of P

BLind-v1-amo-hao-nirS.

Build

Obtained and verified the P

BLind-v1-amo-hao-nirS-nar

as shown in

Fig. 1.7A.

According to the restriction endonuclease sites, used the corresponding restriction enzymes to digest gene cluster and pHY-P43 vector, where the digested pHY-P43 vector was shown in

Fig. 1.7B.

After digestion, we used T4 DNA ligase to ligate P

BLind-v1-amo-hao-nirS

and pHY-P43, and then transferred it into DH5α. The transformation plate was named I and the plasmid was named pHY-P43/P

BLind-v1-amo-hao-nirS.

Transferred it into Bs/WT and the transformation plate was named II, and the plasmid was named pHY-P43-P

BLind-v1-amo-hao-nirS.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-7.png)

Fig. 1.7

(A) Lane 1: DNA Marker, Lane2: P

BLind-v1-amo-hao-nirS

gene fragment, amplified by rTaq DNA polymerase at annealing temperature at 63 ℃, and the band size was as expected. (B) Lane 1: DNA Marker, Lane 2: pHY-P43 vector digested with

Hind

Ⅲ and

BamH

Ⅰ, and the band size was as expected.

Test

The colonies were selected from the Ⅰ and inoculated into the medium containing Tetracycline for overnight culture. Extracted the plasmid and carried out verification of the size of pHY-P43-P

BLind-v1-amo-hao-nirS

and verification of the P

BLind-v1-amo-hao-nirS.

The plasmid verification results were shown in

Fig. 1.8.

The colonies were selected from the Ⅱ and inoculated into the medium containing Tetracycline for overnight culture. Cultivated till OD

600=1.0, transferred 100 μL to 5 mL of new medium containing Tetracycline, meanwhile, set a control group and inoculated 100 μL Bs/WT to 5 mL of medium containing Tetracycline. The results of overnight incubation were shown in

Fig. 1.9.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-8.png)

Fig. 1.8

Validation of pHY-P43-P

BLind-v1-amo-hao-nirS.

(A) Comparison of plasmid size between pHY-P43 and pHY-P43-P

BLind-v1

-amo-hao-nirS,

Lane 1: pHY-P43(4.3 kb), Lane 2: pHY-P43-P

BLind-v1-amo-hao-nirS

(7.3 kb). (B) Validation of P

BLind-v1-amo-hao-nirS

used pHY-P43-P

BLind-v1-amo-hao-nirS

as the template, Lane 1: DNA Marker, Lane 2: P

BLind-v1-amo-hao-nirS.

![]( https://static.igem.wiki/teams/4354/wiki/results/result1-9.png)

Fig. 1.9

The left: Bs/pHY-P43-P

BLind-v1-amo-hao-nirS,

The right: Bs.

Learn

Fig. 1.8

showed that we have successfully cloned the gene cluster into pHY-P43.

Fig. 1.9

showed that the we have successfully obtained Bs/pHY-P43-P

BLind-v1-amo-hao-nirS

strains. This meant that we can use blue light to regulate and reduce the nitrogen content in sewage theoretically. Next, we needed to construct the blue light switch.

### 2. Construction the switch of blue light metabolic pathway

The goal of our project requires blue light to regulate the transcription of our candidate genes, and we have successfully constructed the candidate genes expression system based on blue light regulation. Next, we needed to construct our switch device. According to the references and the project of NUS 2021 iGEM team, we selected EL222 as the photosensitive protein of blue light which derived from the light-operated transcription factor of Marine bacterium

Erythrobacter litoralis

HTCC2594, whose N-terminal LOV (light-oxygen-voltage) domain responsible for photosensitivity is linked to the C-terminal HTH (helix-turn-helix) DNA-binding domain. Under dark conditions, the HTH domain was closed by LOV. Upon blue light irradiation, the conformation of the LOV domain changes and the HTH domain is released, and the protein dimerizes and binds to the specific DNA sequence to initiate gene transcription. The detailed design can be referred to

Fig. 2.1.

![]( https://static.igem.wiki/teams/4354/wiki/results/result2-1.png)

Fig. 2.1

Design and characterization of the blue light inducible gene expression system.

Design

Cloned P43

-el222

into pUC19 to obtain high expression level photosensitive protein shown in

Fig. 2.2.

pUC19 with low molecular weight but high copy number.

![]( https://static.igem.wiki/teams/4354/wiki/results/result2-2.png)

Fig. 2.2

The construction of P43

-el222.

Build

Obtained and verified the P43

-el222

Fig. 2.3A.

According to the restriction endonuclease sites, used the corresponding restriction enzymes to digest P43

-el222

and pUC19, where the digested pUC19 vector was shown in

Fig. 2.3B.

After digestion, we used T4 DNA ligase to ligate P43-

el222

and pUC19, and then transferred it into DH5α followed by verification.

![]( https://static.igem.wiki/teams/4354/wiki/results/result2-3.png)

Fig. 2.3

(A) Lane 1: DNA Marker, Lane 2: P43-

el222

gene fragment, amplified by rTaq DNA polymerase at annealing temperature at 61 ℃, and the band size was as expected. (B) Lane 1: DNA Marker, Lane 2: pUC19 vector digested with

Pci

Ⅰ and

Xba

Ⅰ, the band size was as expected.

Test

The colonies were selected from the transformation plate and inoculated into medium containing Ampicillin for overnight culture. Extracted the plasmid and carried out verification of the size of pUC19-P43-

el222

and verification of the P43-

el222.

The plasmid verification results were shown in

Fig. 2.4.

![]( https://static.igem.wiki/teams/4354/wiki/results/result2-4.png)

Fig. 2.4

Validation of pUC19-P43

-el222.

(A) Comparison of plasmid size between pUC19 and pUC-P43

-el222,

Lane 1: pUC19 (2.3 kb), Lane 2: pUC19-P43

-el222

(3.4 kb). (B) Validation of P43

-el222

used pUC19-P43

-el222

as the template, Lane 1: DNA Marker, Lane 2: P43

-el222.

Learn

Fig. 2.4

showed that we have successfully cloned P43

-el222

into pUC19, which will be used for starting the expression of gene cluster, laying the foundation for the success of the subsequent blue light pathway.

### 3. Detection of candidate genes expression Detection of nar expression

#### Detection of nar expression

Design

We added the

gfp

(the gene for coding green fluorescent protein) obtained from ZJU-Ghina to show the expression level of nar, shown in

Fig. 3.1.

![]( https://static.igem.wiki/teams/4354/wiki/results/result3-1.png)

Fig. 3.1

The construction of P43

-nar-gfp.

Build

Amplified

gfp

from the pHY300PLK-P

veg-gfp

which obtained from ZJU-China, shown in

Fig. 3.2.

Sent the

gfp

and our successfully constructed pHY-P43

-nar

to BGI company for integration to ensure that nar and

gfp

are a fusion protein. Then, transferred pHY-P43

-nar-gfp

and pHY-P43

-nar

into Bs/WT separately.

![]( https://static.igem.wiki/teams/4354/wiki/results/result3-2.png)

Fig. 3.2

Lane 1: DNA Marker, Lane 2:

gfp

gene fragment, amplified by rTaq DNA polymerase at annealing temperature at 61 ℃, and the band size was as expected.

Test

The colonies were selected from the transformation plate and inoculated into medium containing Tetracycline for overnight culture. The results showed in

Fig. 3.3.

And transferred the strains that can grow in Tetracycline containing culture medium until OD

600

=1.0, and detect the fluorescence intensity of GFP by qPCR to show the expression level of nar. The result was shown in

Fig. 3.4.

![]( https://static.igem.wiki/teams/4354/wiki/results/result3-3.png)

Fig 3.3

(A) The left: Bs/pHY-P43

-nar-gfp,

The right: Bs/WT. (B) The left: Bs/pHY-P43

-nar,

The right: Bs/WT.

![]( https://static.igem.wiki/teams/4354/wiki/results/result3-4.png)

Fig. 3.4

Determination of fluorescence intensity of GFP. Four asterisks (\*\*\*\*) in the figure denote a significant difference (P< 0.0001, two-tailed Student's t-test) between two groups. Error bars represent the standard deviation (SD) of the data derived from three biological replicates.

-nar-gfp,

The right: Bs/WT. (B) The left: Bs/pHY-P43

-nar,

The right: Bs/WT.

Learn

Fig. 3.3

showed that we have successfully obtained Bs/pHY-P43

-nar-gfp

and Bs/pHY-P43

-nar

strains.

Fig. 3.4

showed that the strong fluorescence signal was successfully detected. This demonstrates that

nar

was expressed successfully in the strains of Bacillus subtilis, which would be beneficial to our treatment of nitrate in sewage.

#### Detection of amo-hao-nirS expression

Design

Used the blue light of the hardware group to irradiate the bacterial liquid of Bs/pHY-P43-P

BLind-v1-amo-hao-nirS/

pUC19-P43

-el222

strains, and then detected the transcription of gene cluster by RT-qPCR.

Build

Transferred pUC19-P43

-el222

into Bs/pHY-P43-P

BLind-v1-amo-hao-nirS

and tested through selecting antibiotic resistance makers. The strains that can grow in Tetracycline and Ampicillin containing were divided into two groups and transferred into new culture medium to culture to OD

600

=1.0, and then one group was irradiated with 450 nm blue light for 12 hours under 37 ℃, the other group was placed under the same conditions but kept away from light. Then RNA was extracted from two sets for RT-qPCR.

Test

The results of resistance screening experiment showed in

Fig. 3.5.

The results of RT-qPCR showed in

Fig. 3.6.

![]( https://static.igem.wiki/teams/4354/wiki/results/result3-5.png)

Fig. 3.5

The left: Bs/pHY-P43-P

BLind-v1-amo-hao-nirS/

pUC19-P43

-el222,

The right: Bs/pHY-P43-P

BLind-v1-amo-hao-nirS.

![]( https://static.igem.wiki/teams/4354/wiki/results/result3-6.png)

Fig. 3.6

Results of RT-qPCR for each gene in the gene cluster. The experimental group was irradiated with 450 nm blue light for 12 hours. Asterisks represent significant difference (\*\*\*\*, P < 0.0001; ns, not significant, two-tailed Student's t-test) between two groups. Error bars represent the standard deviation (SD) of the data derived from three biological replicates.

Learn

Fig. 3.5

showed that we have successfully obtained Bs/pHY-P43-P

BLind-v1-amo-hao-nirS/

pUC19-P43

-el222

strains. Compared the control without the blue light induction, the transcription level of the gene cluster was significantly up regulated as shown in

Fig. 3.6.

This also meant that blue light can induce a significant increase in the level of candidate gene expression, which was in line with our expected results. So far, our blue light gene expression systems have been constructed, and their expression can be detected in Bs/WT. This plays an important role in our project, light regulated and sewage treatment.

### 4. Construction of candidate genes expression vector based on green light regulation

Too low total nitrogen in water or too high density of our engineered bacteria in water will pose potential threats to the surrounding environment. Therefore, we designed a mechanism to enable the host to express nuclease through green light regulation to achieve host suicide by referring to the literature as shown

Fig. 4.1.

Under green light irradiation, CcaS phosphorylates itself, and then the phosphate group transfers to the response regulator CcaR. The phosphorylated CcaR forms a dimer and combines with the P

cpcG2-veg,

and the expression of the candidate gene is started. We selected

nucA

(for coding NucA, which is an extracellular enzyme that can hydrolyze nucleic acid) as our candidate gene, and used P

cpcG2-veg

to started its transcription.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-1.png)

Fig. 4.1

Bacillus subtilis

schematic diagram of green light regulation principle. (A) The construction of control green light switch. (B) The construction of P

cpcG2-veg-nucA.

#### Cloned the candidate gene regulated by green light to Bs/pHY-P43-P BLind-v1-amo-hao-nirS /pUC19-P43 -el222

Design

Considering the length of the sequence and the possibility of plasmid loss when multiple plasmids were transferred into the host, we constructed P

cecG2-veg-nucA

downstream of P43

-el222

in pUC19-P43

-el222

shown in

Fig. 4.2.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-2.png)

Fig. 4.2

The construction of P

cecG2-veg-nucA.

Build

Obtained and verified the P

cecG2-veg-nucA.

as shown

Fig. 4.3A.

According to the restriction endonuclease sites, selected the corresponding restriction enzymes to digest P

cecG2-veg-nucA

and our constructed previously pUC19-P43

-el222,

where the digested pUC19-P43

-el222

vector was shown in

in Fig. 4.3B.

After digestion, we used T4 DNA ligase to ligate P

cecG2-veg-nucA

and pUC19-P43

-el222,

and then transfer it into DH5α, the transformation plate was named as Ⅰ and the plasmid was named pUC19-P43

-el222

-P

cecG2-veg-nucA

and transferred it into our previously constructed Bs/pHY-P43-P

BLind-v1-amo-hao-nirS,

the transformation plate was named as Ⅱ.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-3.png)

Fig. 4.3

(A) Lane 1: DNA Marker, Lane 2: P

cecG2-veg-nucA

gene fragment, amplified by rTaq DNA polymerase at annealing temperature at 60 ℃, and the band size was as expected. (B) Lane 1: DNA Marker, Lane 2: pUC19-P43

-el222

vector digested with

Xba

I and

EcoR

I, the band size was as expected.

Test

The colonies were selected from the Ⅰ and inoculated into the medium containing Ampicillin for overnight culture. Extracted the plasmid and carried out verification of the size of pUC19-P43

-el222-

P

cecG2-veg-nucA

and verification of the P

cecG2-veg-nucA.

The plasmid verification results were shown in

Fig. 4.4.

The colonies were selected from the Ⅱ and inoculated into the medium containing Tetracycline and Ampicillin for overnight cultivate till OD

600

=1.0, transferred 100 μL to 5 mL of new medium containing Tetracycline, meanwhile, set a control group and inoculated 100 μL Bs/pHY-P43-P

BLind-v1-amo-hao-nirS

to 5 mL of medium containing Tetracycline and Ampicillin. The results of overnight incubation were shown in

Fig. 4.5.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-4.png)

Fig. 4.4

Validation of pUC19-P43

-el222

-P

cpcG2-veg-nucA.

(A) Comparison of plasmid size between pUC19-P43

-el222

(3.4 kb) and pUC-P43

-el222

-P

cpcG2-veg-nucA.

(B) Validation of P

cpcG2-veg-nucA

used pUC19 -P43-el222-P

cpcG2-veg-nucA

as the template, Lane 1: DNA Marker, Lane 2: P43

-el222,

Lane 3: P

cpcG2-veg-nucA.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-5.png)

Fig. 4.5

The left: Bs/pHY-P43-P

BLind-v1-amo-hao-nirS/

pUC19-P43

-el222

-P

cpcG2-veg-nucA,

The right: Bs/pHY-P43-P

BLind-v1-amo-hao-nirS.

Learn

Fig. 4.4

showed that we have successfully cloned P

cpcG2-veg-nucA

into pUC19-P43

-el222,

which had laid a solid foundation for the subsequent realization of blue-green light regulation.

Fig. 4.5

showed that we have successfully obtained Bs/pHY-P43-P

BLind-v1-amo-hao-nirS/

pUC19-P

cpcG2-veg-nucA,

which is very important for our project and makes it possible for us to control the bacterial density in time, maintain the nitrogen content in the water body and maintain the orderly operation of the ecosystem.

#### Cloned the candidate gene regulated by green light to Bs/pHY-P43 -nar

Design

nar

was not connected with the blue light inducible promoter P

BLind-v1,

and considered the length of the sequence and the possibility of plasmid loss when multiple plasmids were transferred into the host, and we constructed P

cpcG2-veg-nucA

in pUC19, such as

Fig. 4.6,

and then transferred it into pUC19.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-6.png)

Fig. 4.6

The construction of P

cpcG2-veg-nucA.

Build

Obtained and verified the P

cpcG2-veg-nucA

shown

Fig. 4.7A.

According to the restriction endonuclease sites, selected the corresponding restriction enzymes to digest P

cpcG2-veg-nucA

and pUC19, where the digested pUC19 vector was shown

Fig. 4.7B.

After digestion, we used T4 DNA ligase to ligate P

cpcG2-veg-nucA

and pUC19, and then transferred it into DH5α, the transformation plate was named as Ⅰ and the plasmid was named pUC19-P

cpcG2-veg-nucA,

and transferred it into our previously constructed Bs/pHY-P43-

narz,

the transformation plate was named as Ⅱ.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-7.png)

Fig. 4.7

(A) Lane 1: DNA Marker, Lane 2: P

cpcG2-veg-nucA,

amplified by rTaq DNA polymerase at annealing temperature at 60 ℃, and the band size was as expected. (B) Lane 1: DNA Marker, Lane 2: pUC19 vector digested with

Xba

Ⅰ and

EcoR

Ⅰ, and the band size was as expected.

Test

The colonies were selected from the Ⅰ and inoculated into the medium containing Ampicillin for overnight culture. Extracted the plasmid and carried out verification of the size of pUC19-P

cpcG2-veg-nucA.

The plasmid verification results were shown in

Fig. 4.8.

The colonies were selected from the Ⅱ and inoculated them into the medium containing Tetracycline and Ampicillin for overnight culture till OD

600

=1.0, transferred 100 μL to 5 mL of new medium containing Tetracycline, meanwhile, set a control group and inoculated 100 μL Bs/pHY-P43

-nar

to 5 mL of medium containing Tetracycline and Ampicillin. The results of overnight incubation showed in

Fig. 4.9.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-8.png)

Fig. 4.8

Validation of pUC19-P

cpcG2-veg-nucA.

(A) Comparison of plasmid size between pUC19 (2.7 kb) and pUC19-P

cpcG2-veg-nucA

(3.2 kb), Lane 1: pUC19, Lane 2: pUC19-P

cpcG2-veg-nucA.

(B) Validation of P

cpcG2-veg-nucA

used pUC19-P

cpcG2-veg-nucA

as the template, Lane 1: DNA Marker, Lane 2: P

cpcG2-veg-nucA.

![]( https://static.igem.wiki/teams/4354/wiki/results/result4-9.png)

Fig. 4.9

The left: Bs/pHY-P43

/

-nar

pUC19-P

cpcG2-veg-nucA,

The right: Bs/pHY-P43

-nar.

Learn

Fig. 4.8

showed that we have successfully cloned P

cpcG2-veg-nucA

into pUC19,

Fig. 4.9

showed that we have successfully obtained Bs/pHY-P43

-nar/

pUC19-P

cpcG2-veg-nucA

strains, which provides the possibility for us to use green light to control the bacterial density in time, maintain the nitrogen content in the water body and maintain the orderly operation of the ecosystem.

### 5. Construction the switch of green light metabolic pathway

According to the references, we selected the appropriate green light pathway to regulate the expression of nuclease hydrolase (NucA), the green light switch including the green/red light reversible two-component system CcaSR, and two metabolic enzymes for the production of chromophore phycobiliprotein (PCB), such as

Fig. 5.1.

![]( https://static.igem.wiki/teams/4354/wiki/results/result5-1.png)

Fig 5.1

Bacillus subtilis

schematic diagram of green light regulation principle.

This system has been successfully used in Bs/WT. For example, Dr. Tabor’s group has used it for their study. In early May, we contacted Jeffrey J. Tabor's research group to obtain the strain. Due to the epidemic situation and logistics, we failed to obtain the strain. To this end, we began to look for research groups that had used the strain in China, hoping to get their help. After many twists and turns, in the middle of June we finally learned that the green light pathway was used in a graduate student’s research in China. Unfortunately, due to force majeure factors such as epidemic control, the strain could not be transported through logistics, and failed return after many attempts. However, as the competition deadline is approaching, we can only reluctantly give up the construction of the green light switch, but we never give up the consideration of biological safety, and will continue to improve in the future.

In general, we successfully introduced nucleic acid hydrolases into

Bacillus subtilis,

as well as four catalytic enzymes in the denitrification pathway. Three enzymes, including NIR which is the nitrogen removal restriction enzyme, were regulated by blue light, and the nucleic acid hydrolases were regulated by green light.

* [1. Construction of expression system of candidate genes involved in nitrogen removal](#tabs-1-1)
* [2. Construction the switch of blue light metabolic pathway](#tabs-1-2)
* [3. Detection of candidate genes expression](#tabs-1-3)
* [4. Construction of candidate genes expression vector based on green light regulation](#tabs-1-4)
* [5. Construction the switch of green light metabolic pathway](#tabs-1-5)

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