Team:SZPT-CHINA/Engineering
Engineering
Overview
Engineering Success
BBa_K3740050
Design
Strategy
Experimental Results
BBa_K3740030
Design
Strategy
Experimental Results
Learning
BBa_K3740044
Design
Strategy
Experimental Results
Reference
● Overview：
The goal of project is to use optogenetically engineered Gluconacetobacter
hansenii to
fight
with Pseudomonas aeruginosa infection and produce a bacterial cellulose dressing
simultaneously. The
genetic engineering consists of three modules, antipseudomonal drug production module, c-di-GMP
signaling and BC film production module, safety and drug release module.
We have so far made a lot of progress in this project. Hereby, we presented a summary of our
engineering
data. Although this project is still ongoing and improvements are needed to make in some sections,
we
believe that our work can contribute to the iGEM community.
● Engineering Success：
I. Antipseudomonal drug production module（BBa_K3740050）
1. Design
We designed an antipseudomonal drug production module to specifically combat P.
aeruginosa
infection. The receptor domain, the translocase domain of pyocin S2 and the nuclease domain of
colicin
E3
are fused together to get a new chimeric bacteriocin termed SE. The antipseudomonal SE protein can
target and inhibit P. aeruginosa, even including those harbouring a S2 immunity protein
IMMS2. IMME3
is
an immunity protein derived from Escherichia coli. It can bind to the nuclease domain of SE
specifically
and inhibit its activity, thus rendering the host cell immune to SE. A similar expression level of
the
antipseudomonal protein SE and the immunity protein IMM is designed to be expressed in the
engineered
bacteria, thus preventing the bactericidal effect of SE protein on the bacterial chassis itself.
2. Strategy
Two plasmids S2-PET28A and SE-PET28A were initially constructed and introduced into
E.
coli
BL21. Following the induction of lysis protein by IPTG, bacterial cells were in turn disrupted and
the
supernatant containing the soluble SE proteins was collected. The molecular weight of SE protein was
checked by SDS-PAGE while its specificity to P. aeruginosa was also verified using the drop
plate
assay;
In order to improve the antipseudomonal effect, we engineered the RBS to modulate its strength in
controlling SE protein expression. Finally, we identified pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrnB
T1(BBa_K3740050) as our
optimal genetic circuit.
Figure 1. The gene circuit of pR-RBS300-SE-B1006-J23118-RBSII-IMM- rrnB T1
(BBa_K3740050)
3. Experimental results:
3.1 Sequencing
Plasmids from E. coli cells were extracted and sequenced. Analysis based on
sequencing
data indicated that the SE-encoded sequence and pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrnB T1
(BBa_K3740050) have been
successfully integrated into the plasmid vectors.
3.2 Characterization of fusion protein SE
3.2.1 SDS-PAGE
Method: IPTG was added to a final concentration of 0.1 mmol/mL to induce SE protein
expression in E. coli BL21 and subsequently incubated at 25℃ for 12 hours. Supernatant
containing SE
or
S2 proteins was obtained by sonication and centrifugation respectively. The crude protein extract
was
loaded into SDS-PAGE gel and stained by Coomassie Blue to verify the presence of target proteins
(For
more detailed steps, please click Experiments).
After induction by IPTG, S2 and SE protein expression in S2-PET28A-BL21 (lane 2) and SE-PET28A-BL21 (lane 3) has been identified. By contrast, target proteins were not expressed in the strain with S2-PET28A-BL21 (lane 1) and SE -PET28A-BL21 (lane 4) in the absence of IPTG, indicating that expression of S2 and SE proteins can be successfully induced in E. coli transformed with S2-PET28A and SE-PET28A, respectively.
After induction by IPTG, S2 and SE protein expression in S2-PET28A-BL21 (lane 2) and SE-PET28A-BL21 (lane 3) has been identified. By contrast, target proteins were not expressed in the strain with S2-PET28A-BL21 (lane 1) and SE -PET28A-BL21 (lane 4) in the absence of IPTG, indicating that expression of S2 and SE proteins can be successfully induced in E. coli transformed with S2-PET28A and SE-PET28A, respectively.
Figure 2. SDS-PAGE analysis of protein expression in engineered E. coli BL21.
Lane 1,
S2-PET28A-BL21 without IPTG induction; Lane 2, S2-PET28A-BL21 with induction by 0.1mmol/mL IPTG;
Lane 3,
SE-PET28A-BL21 with induction by 0.1mmol/mL IPTG; Lane 4, SE-PET28A-BL21 without IPTG induction.
3.2.2 SE protein targeting verification
Method: Supernatant containing SE or S2 protein was respectively mixed with the culture
of
Pseudomonas aeruginosa PAO1, P. aeruginosa PAO1Δ1150-1151(PAO1 Δ1150-1151, PAO1
knocked out
of
S2+IMMS2)
and E. coli MG1655. PBS was used as a negative control. Inhibitory effect of supernatant on
the P.
aeruginosa growth was determined by measuring OD600 using SYNERGY H1 micro-plate
reader. (For more
detailed steps, please click Experiments)
We found that the OD600 values of both P. aeruginosa strains cultured with SE protein, were much lower than that of the control group PBS. This proves that SE protein has antipseudomonal properties. In contrast, pyocin S2 could only act on the immune-deficiency strain PAO1∆1150-1151. This proves that SE protein has a broader antipseudomonal spectrum than S2. Meanwhile, the OD600 value of E. coli MG1655 cultured with SE protein was a little higher than that of the control group PBS. This indicates that SE protein does not work on E. coli. This result indicated that the fusion protein SE can specifically kill P. aeruginosa.
We found that the OD600 values of both P. aeruginosa strains cultured with SE protein, were much lower than that of the control group PBS. This proves that SE protein has antipseudomonal properties. In contrast, pyocin S2 could only act on the immune-deficiency strain PAO1∆1150-1151. This proves that SE protein has a broader antipseudomonal spectrum than S2. Meanwhile, the OD600 value of E. coli MG1655 cultured with SE protein was a little higher than that of the control group PBS. This indicates that SE protein does not work on E. coli. This result indicated that the fusion protein SE can specifically kill P. aeruginosa.
Figure 3. Optical density of bacterial culture of PAO1, PAO1 Δ1150-1151 and E.
coli
MG1655
treated by the supernatant of S2-PET28A-BL21 or SE-PET28A-BL21 for 790 minutes. PBS was used as the
negative control.
3.3 Batch screening and verification of SE protein antibacterial performance of
composite
parts
Method: PAO1 growth inhibition verification. G. hansenii ATCC 53582 culture
supernatant
containing SE proteins that were expressed under control of different RBS were obtained by
sonication
and centrifugation and further purified by infiltration with micro-pored membrane. Then, supernatant
in
different groups was individually mixed with PAO1 culture, and incubated at 30℃ for 12 hours.
SYNERGY H1
micro-plate reader was used to measure the OD600 to determine the effect on PAO1 growth.
Inhibition zone experiment. 3μL of the supernatant containing SE protein was dropped on FAB
plate
with
PAO1（For more detailed steps, please click Experiments).
As shown in Figure 4 (a), we found that the OD600 values of PAO1 strains cultured with supernatant of the 3rd, 4th, 7th, and 8th colonies bacterial lysate, were much lower than that of PBS in the control group. This proves that culture supernatant of the 3rd, 4th, 7th, and 8th colonies had a remarkable inhibitory effect on the growth of PAO1. Consistent with this result, supernatants from all the G. hansenii isolates have formed inhibition zone on the plate spread with PAO1, yet with different sizes. The 3rd and 4th isolate displayed the most significant antipseudomonal effect, while the control, the 9th colony had no effect. Taken together, these results show that expression of SE protein was successfully induced in G. hansenii ATCC 53582. Besides, by adjusting the RBS upstream the SE-encoded sequence, we screened and identified that the 4th strain, pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrnB T1-pSEVA331-G. hansenii ATCC 53582-4# exhibited the optimal antipseudomonal effect.
As shown in Figure 4 (a), we found that the OD600 values of PAO1 strains cultured with supernatant of the 3rd, 4th, 7th, and 8th colonies bacterial lysate, were much lower than that of PBS in the control group. This proves that culture supernatant of the 3rd, 4th, 7th, and 8th colonies had a remarkable inhibitory effect on the growth of PAO1. Consistent with this result, supernatants from all the G. hansenii isolates have formed inhibition zone on the plate spread with PAO1, yet with different sizes. The 3rd and 4th isolate displayed the most significant antipseudomonal effect, while the control, the 9th colony had no effect. Taken together, these results show that expression of SE protein was successfully induced in G. hansenii ATCC 53582. Besides, by adjusting the RBS upstream the SE-encoded sequence, we screened and identified that the 4th strain, pR-RBS300-SE-B1006-J23118-RBSII-IMM-rrnB T1-pSEVA331-G. hansenii ATCC 53582-4# exhibited the optimal antipseudomonal effect.
Figure 4. (a) Optical density of bacterial culture of PAO1 treated by the supernatant of
engineered strains for 790 minutes. (b) Inhibition zone test. (c) Strains are used for Figure(a) and
Figure(b).
3.4 Video shooting
Video 1. Video shooting of the zone of inhibition of 4th bacteria:
pR-RBS300-SE-J23118-RBSII- IMM-rrnB T1-pSEVA331-G. hansenii ATCC 53582-4#.
II. C-di-GMP signaling and BC film production module (BBa_K3740030)
1. Design
This c-di-GMP signaling and BC film production module has two submodules. One is the
diguanylate cyclase sub-module, which regulates the synthesis of c-di-GMP. The bacterial phytochrome
BphS is utilized to synthesize c-di-GMP in a light-dependent manner, thereby controlling BC film
production in G. hansenii ATCC 53582. The other is c-di-GMP phosphodiesterase sub-module,
which
regulates the hydrolysis of c-di-GMP. By screening different parts, the level of c-di-GMP in
bacteria
can remain in a low level or even close to zero under the dark condition.
2. Strategy
A series of plasmids derived from J23100-B0034 were constructed and transferred into
G.
hansenii ATCC 53582, in which the BC film production was quantified. Finally, fcsR (BBa_K3740022)
was
selected as the c-di-GMP phosphodiesterase encoding gene. Different promoters were then used to
direct
the expression of FcsR. Based on the screening results, BBa_K3740030,
BBa_K3740031 and
BBa_K3740043
were
constructed and introduced into G. hansenii ATCC 53582 through electroporation. Finally, BC
film
production was assessed in these strains.
Figure 5. The gene circuit of J23100-B0034-bphS-pET RBS-bphO-rrnB T1-J23109-B0034-fcsR-
rrnB
T1 (BBa_K3740030).
3. Experimental results
3.1 Agarose gel electrophoresis
As shown in Figure 6 (a), c-di-GMP phosphodiesterase-encoded genes (BBa_K3740065),
(BBa_K3740062)
and (BBa_K3740066) were
identified successfully by PCR amplification and c-di-GMP diguanylate
cyclase-encoded genes (BBa_K3740047) was
identified by PCR amplication; As shown in Figure 6(b),
composite
parts including (BBa_K3740030), (BBa_K3740031)
and (BBa_K3740043) were
identified successfully by PCR.
Figure 6. (a), (b)The agarose gel electrophoresis image of the constructed plasmid after
PCR
amplification. (c)Plasmids are used for Figure(a) and Figure(b).
3.2 Film production verification experiment
3.2.1 Film production verification experiment of bphS and fcsR
Method: The constructed plasmids were introduced into G. hansenii ATCC 53582 and
cultured at
30°C. Then a single colony was picked to launch its liquid culture. Then the culture was distributed
in
two copies of 12-well plates, with one under NIR light illumination and the other under dark
condition
for 4 days. (For more detailed steps, please click Experiments)
As shown in Figure 7 (a), BC production in J23100-fcsR-rrnB T1-pSEVA331- G. hansenii ATCC 53582 and the control group pSEVA331- G. hansenii ATCC 53582 were different, indicating that FcsR was capable of hydrolyzing c-di-GMP in G. hansenii. While, J23100-bphS-pET RBS-bphO-rrnB T1-pSEVA331-G. hansenii ATCC 53582, produced a higher level of BC film under near-infrared light than that under dark condition, indicating that BphS in G. hansenii ATCC 53582 can synthesize c-di-GMP. To summarize, BphS and FcsR were opted as the c-di-GMP synthetase and hydrolase in our system, respectively.
As shown in Figure 7 (a), BC production in J23100-fcsR-rrnB T1-pSEVA331- G. hansenii ATCC 53582 and the control group pSEVA331- G. hansenii ATCC 53582 were different, indicating that FcsR was capable of hydrolyzing c-di-GMP in G. hansenii. While, J23100-bphS-pET RBS-bphO-rrnB T1-pSEVA331-G. hansenii ATCC 53582, produced a higher level of BC film under near-infrared light than that under dark condition, indicating that BphS in G. hansenii ATCC 53582 can synthesize c-di-GMP. To summarize, BphS and FcsR were opted as the c-di-GMP synthetase and hydrolase in our system, respectively.
Figure 7. (a)BC yield by J23100-B0034-fcsR-rrn B T1-pSEVA331-G. hansenii ATCC
53582,
J23100-B0034-yhjH-rrnB T1-pSEVA331-G. hansenii ATCC 53582, J23100-B0034-rocR-rrnB
T1-pSEVA331-G.
hansenii
ATCC 53582 and the vehicle control pSEVA331-G. hansenii ATCC 53582; (b) BC yield by
J23100-bphS-pET
RBS-bphO-rrnB T1-pSEVA331-G. hansenii ATCC 53582 under NIR light illumination and dark
condition.
3.2.2 Film production verification experiment of bphS-fcsR
A significant difference in BC film production under NIR light and dark conditions were
observed for those strains, including J23100-B0034-bphS-pET RBS-bphO-rrnB T1-J23109-B0034-fcsR-rrnB
T1-pSEVA331-G. hansenii ATCC 53582, J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23110-B0034-fcsR-rrnB
T1-pSEVA331-G. hansenii ATCC 53582, however J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23119-B0034-fcsR-rrnB
T1-pSEVA331-G. hansenii ATCC 53582 had no difference in BC film production under NIR light
and dark
conditions, these results showed that J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23109-B0034-fcsR-rrnB
T1-pSEVA331-G. hansenii ATCC 53582 and J23100-B0034-bphS-pET RBS-bphO-rrnB
T1-J23110-B0034-fcsR-rrnB
T1-pSEVA331-G. hansenii ATCC 53582 can be used as our gene circuit.
Figure 8. (a) BC film production by different strains, (b) Strains are used for
Figure(a).
3.3 Video shooting
Video 2. Video shooting of J23100-B0034-bphS-pET RBS-bphO-rrnB T1-J23109-B0034-fcsR-rrnB
T1-pSEVA331-G. hansenii ATCC 53582 bacterial BC production.
4. Learning:
In our film production assay, the film production by engineered bacteria under dark
condition was expected to remain at a very low level. Surprisingly, a substantial amount of BC film
was
still found without NIR light induction, even though the level was lower than that in the
NIR-induced
group. In the future, we will solve this problem and refine the genetic circuit.
III. Safety and drug release module (BBa_K3740044)
1. Design
This module is a dual design of both safety control and drug release. We use pDawn, a
blue-light-responsive promoter to direct the expression of the lytic proteins, which would cause the
lysis
and death of the engineered bacteria. Then, SE protein expressed within the engineered bacteria will
be
released.
We have constructed a set of plasmids which include pDawn to induce the expression of an
anti-holin-free
Lambda lysis cassette (S105), φX174 lysis gene (X174 E), and LKD16 lysis cassette (LKD16). They were
chosen
to lyse our chassis bacteria. After screening, X174 E (BBa_K2656015) was
selected as the final part
in
our
safety and drug release module.
2. Strategy
RFP was firstly used to verify the responsiveness of the pDawn promoter to blue light.
Then,
random primer guided mutagenesis method to modulate the strength of the RBS (BBa_B0034) located
upstream
of
X174 E. Finally, the pDawn-RBSNNN-X174 E-rrnB T1 (BBa_K3740044) plasmid
was constructed and
introduced
into
E. coli. E. coli isolates that grow normally in the dark and cannot grow under blue
light
were
screened
out.
Finally, the plasmid was extracted, and further introduced into G. hansenii ATCC 53582, in
which the
responsiveness of pDawn to blue light was also verified.
Figure 9. The gene circuit of pDawn-RBSNNN-X174 E-rrnB T1 (BBa_K3740044).
3.Experimental results
3.1 The pDawn promoter responds to blue light verification in G. hansenii ATCC
53582
Red fluorescence could be observed in the pDawn-B0034-RFP-rrnB T1-pSEVA331-G.
hansenii
ATCC
53582 under 470-nm blue light, but there was no red fluorescence under dark condition,
suggesting
that
the pDawn promoter could respond to blue light and induce gene expression in G. hansenii
ATCC
53582.
Figure 10. Red fluorescence photo of the pDawn-B0034-RFP-rrnB T1-pSEVA331-G.
hansenii
ATCC
53582 cultured under 470-nm blue light (a) or dark condition (b) for 24 hours.
3.2 Batch screening of pDawn-RBSNNN- X174 E-rrnB T1-pSEVA331 in response to blue light
lysis
in E. coli
Method: We use the random primer method to modulate the strength of the RBS (BBa_B0034) located
upstream
of X174 E. After introducing into E. coli DH5α, a drop plate assay was performed to screen
the
bacterial
isolate that can grow normally in the dark but cannot under the blue light irradiation.
As shown in Figure 11, the 4th isolate that grew normally in the dark but did not under blue light, indicating that we successfully expressed the cleavage protein X174 E in E. coli.
As shown in Figure 11, the 4th isolate that grew normally in the dark but did not under blue light, indicating that we successfully expressed the cleavage protein X174 E in E. coli.
Figure 11：The growth status of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-DH5α under blue
light
(a)
and dark condition (b).
3.3 pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-7# in response to blue photolysis in G.
hansenii
ATCC 53582
Method: We launched the liquid culture of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-DH5α,
extracted the plasmid and introduced into G. hansenii ATCC 53582. After 2 days of incubation,
we
selected 20 monoclonal to spot on two parallel new plates, which were incubated for another 2 days,
with
one under blue light and the other in the dark. The strains with obvious growth difference under
different illumination conditions were preserved. The drop plate assay using this strain was
repeated to
verify the lysis effect of X174 E on G. hansenii ATCC 53582.
As shown in Figure 12 (a), G. hansenii ATCC 53582 strains under the dark condition exhibited better growth than those under blue light irradiation; (b) pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-G. hansenii ATCC 53582-7# showed a stable lysis effect under blue light illumination but not in the dark, indicating that we successfully expressed the cleavage protein X174 E in G. hansenii ATCC 53582.
As shown in Figure 12 (a), G. hansenii ATCC 53582 strains under the dark condition exhibited better growth than those under blue light irradiation; (b) pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-G. hansenii ATCC 53582-7# showed a stable lysis effect under blue light illumination but not in the dark, indicating that we successfully expressed the cleavage protein X174 E in G. hansenii ATCC 53582.
Figure 12. (a) The growth status of pDawn-RBSNNN-X174 E-rrnB T1-pSEVA331-G.
hansenii ATCC
53582-4# under blue light (1) and dark conditions (2), (b)The growth status of pDawn-RBSNNN-X174
E-rrnB
T1-pSEVA331-G. hansenii ATCC 53582-7# under blue light (1) and dark conditions (2).
In our safety and drug release module, we verified the lysis effect of (BBa_K3740024) and
(BBa_K3740032), however
their effect was inferior to that of (BBa_ K2656015). If you
are interested in
our
other two parts, please Click: (BBa_K3740033) and (BBa_K3740051).
Reference
[1] Gupta S, Bram E E, Weiss R. Genetically programmable pathogen sense and destroy. [J]. Acs
Synthetic
Biology, 2013, 2(12): 715-723.
[2] Jin F, Gao YM, Huang YJ, et al. Current therapies for Pseudomonas aeruginosa infection [J]. Journal of Integration Technology, 2021, 10(4): 50-66.
[3] Michel-Briand Y, Baysse C. The pyocins of Pseudomonas aeruginosa. [J]. Biochimie, 2002, 84(5-6): 499-510.
[4] Denayer S, Matthijs S, Cornelis P. Pyocin S2 (Sa) Kills Pseudomonas aeruginosa Strains via the FpvA Type I Ferripyoverdine Receptor [J]. Journal of Bacteriology, 2007, 189(21): 7663.
[5] Ryu M H, Gomelsky M Near-infrared Light Responsive Synthetic c-di-GMP Module for Optogenetic Applications [J]. Acs Synthetic Biology, 2014, 3(11): 802
[6] Elaheh Sajadi,Seyed Safa-Ali Fatemi,Valiollah Babaeipour,Ali Asghar Deldar,Bagher Yakhchali,Mohammad Saberi Anvar. Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacter xylinus in E. coli Nissle 1917 [J]. Bioprocess and Biosystems Engineering, 2019, 42(12): 2023-2034.
[7] Jin X, Riedel-Kruse I H. Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression [J]. Proceedings of the National Academy of Sciences of the United States of America, 2018: 3698-3703.
[8] Ceyssens P J, Lavigne R, Mattheus W, et al. Genomic Analysis of Pseudomonas aeruginosa Phages LKD16 and LKA1: Establishment of the KMV Subgroup within the T7 Supergroup [J]. Journal of Bacteriology, 2006.
[9] Yibo S, Jianhe S, Shi Y, et al. Current advance in the topological structure and function of holin encoded by bacteriophage lambda [J]. Acta Microbiologica Sinica, 2012, 52(2): 141-145.
[10] Witte A, Wanner G, Sulzner M, et al. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli [J]. Archives of Microbiology, 1992, 157(4): 381-388.
[11] Ohlendorf R, Vidavski R R, Eldar A, et al. From dusk till dawn: one-plasmid systems for light-regulated gene expression [J]. Journal of Molecular Biology, 2012, 416(4): 534-542.
[2] Jin F, Gao YM, Huang YJ, et al. Current therapies for Pseudomonas aeruginosa infection [J]. Journal of Integration Technology, 2021, 10(4): 50-66.
[3] Michel-Briand Y, Baysse C. The pyocins of Pseudomonas aeruginosa. [J]. Biochimie, 2002, 84(5-6): 499-510.
[4] Denayer S, Matthijs S, Cornelis P. Pyocin S2 (Sa) Kills Pseudomonas aeruginosa Strains via the FpvA Type I Ferripyoverdine Receptor [J]. Journal of Bacteriology, 2007, 189(21): 7663.
[5] Ryu M H, Gomelsky M Near-infrared Light Responsive Synthetic c-di-GMP Module for Optogenetic Applications [J]. Acs Synthetic Biology, 2014, 3(11): 802
[6] Elaheh Sajadi,Seyed Safa-Ali Fatemi,Valiollah Babaeipour,Ali Asghar Deldar,Bagher Yakhchali,Mohammad Saberi Anvar. Increased cellulose production by heterologous expression of bcsA and B genes from Gluconacetobacter xylinus in E. coli Nissle 1917 [J]. Bioprocess and Biosystems Engineering, 2019, 42(12): 2023-2034.
[7] Jin X, Riedel-Kruse I H. Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression [J]. Proceedings of the National Academy of Sciences of the United States of America, 2018: 3698-3703.
[8] Ceyssens P J, Lavigne R, Mattheus W, et al. Genomic Analysis of Pseudomonas aeruginosa Phages LKD16 and LKA1: Establishment of the KMV Subgroup within the T7 Supergroup [J]. Journal of Bacteriology, 2006.
[9] Yibo S, Jianhe S, Shi Y, et al. Current advance in the topological structure and function of holin encoded by bacteriophage lambda [J]. Acta Microbiologica Sinica, 2012, 52(2): 141-145.
[10] Witte A, Wanner G, Sulzner M, et al. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli [J]. Archives of Microbiology, 1992, 157(4): 381-388.
[11] Ohlendorf R, Vidavski R R, Eldar A, et al. From dusk till dawn: one-plasmid systems for light-regulated gene expression [J]. Journal of Molecular Biology, 2012, 416(4): 534-542.