Order of constructing plasmids
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
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
.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
