iGEM 2022 — Team cug-china

![](https://static.igem.wiki/teams/4242/wiki/background/b-nb.jpg)

Notebook

* Jan.
* Feb.
* Mar.
* Apr.
* May.
* Jun.
* Jul.
* Aug.
* Sep.
* Oct.

![](https://static.igem.org/mediawiki/2021/7/7e/T--XMU-China--blueleftArrow.png)
![](https://static.igem.org/mediawiki/2021/d/d7/T--XMU-China--bluerightArrow.png)

# Jan.

-Publicized IGEM competition and answered students' questions about iGEM.
 -Recruited team members and interviewed.

# Feb.

-Our team was officially established and held the first meeting!
 -All student members received laboratory safety training.
 -All student members learned the basic concepts and experiments of molecular biology to establish understanding of synthetic biology.

# Mar.

-Brainstorm: Extensively read articles and listed potential topics.
 -Visited Institute of Hydrobiology, Chinese Academy of Sciences to understand the application of synthetic biology.
 -Determined the theme of our team!
 -Assigned tasks to each team.

# Apr.

-Art design group made team logo!
 -All students learned from the previous excellent iGEM projects and reported at the weekly meeting.
 -Designed biobricks.
 -Reached a cooperation intention with XMU-China.

# May.

-Art design group designed each member's cartoon image.
 -Art design group and the dry experiment group learned how to make web together.
 -Continued to learn the projects of previous excellent iGEM teams.
 -Deeply insighted into notions of parts, devices, systems in synthetic biology.

# Jun.

-Participated ARS2022 and shared our project at the conference.
 -Submitted first version of safety form.
 -Constructed the first plasmid pHYD-1 and inserted plasmid pHYD-1 into S.oneidensis MR-1 and E.coli BL21.

# Jul.

-Held online communication with NYCU-formosa to learn their experience in modeling.
 -Collaborated with Xiamen University.
 -Attended Central China iGEM Communication.
 -Constructed plasmid pHYD-2 and inserted plasmid pHYD-2 into chassis.
 -Demonstrate the function of pHYD-2.

# Aug.

-Be present at Conference of China iGEMer Community(CCiC) and introduced our project to other teams.
 -Filmed and made our promotion video.
 -Interviewed CATUG Biotechnology and visited Dr. Zunyang Ke.
 -Constructed plasmid pHYD-3 and codon optimization of fleQ.
 -Transformed plasmid pHYD-3 into BL21 and MR1.
 -Tested the fluorescence using different biofilm dispersal agent.

# Sep.

-Made our homepage!
 -Inserted the pbad into fleQ and did pre-experiment.
 -Utilized HPLC to detect the c-di-GMP level of pHYD-3 strain.
 -Held a joint education conference with HUST-China.
 -Partnership experiments.
 -Transformed the plasmid in to BL21.
 -Adapted the set of ordinary differential equations to better describe the translation and transcription of gene and solved the model by Runge-Kutta method.

# Oct.

-Finish the construciton of team wiki.
 -Documentaion of part improvement.

![](https://static.igem.wiki/teams/4242/wiki/background/background/b-protocols.png)

Engineering

{% include "sider.html" %}

[Overview](#Overview)
[Cycle 1](#Cycle 1)
[Cycle 2](#Cycle 2)
[Cycle 3](#Cycle 3)

# **Silver #1**

# **Overview**

---

In order to connect the c-di-GMP level to the reporter gene, we chose the signaling system FleQ protein from *P. aeruginosa* in which the FleQ protein functions as both a repressor and an activator to control gene expression from the *pel* operon promoter in response to c-di-GMP. However, how to make this system work in other bacteria is a big challenge. We have experienced three engineering cycles to construct a preliminary functional biosensor.

# **Cycle 1: Test the performance of promotor Ppel in *E. coli* and *S. oneidensis***

---

Theoretically, the gene under *pel* promoter can be constitutively expressed in the bacteria that have no repressor FleQ. To test this hypothesis, we directly fused *pel* promoter to *gfp* and we had our first plasmid, pHYD-1. ([BBa\_K4242011](http://parts.igem.org/Part%3ABBa_K4242011)) Then we transferred the biobrick into two module organisms, *Escherichia coli* and *Shewanella oneidensis*. However, no detectable fluorescence was observed, which suggests the promoter Ppel is non-functional in these two bacteria.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/wet-pho/6.png)

Fig. 1 a|Structure of pHYD-1 and control plasmid b|Colony RPC result of pHYD-1

# **Cycle 2: Construct the tandem promotor**

---

To overcome the difficulty we met in cycle1, we designed a tandem promoter PcI/pel in which the transcription start site of the constitutive promoter PcI is immediately followed by the FleQ binding site of the promoter Ppel. ([BBa\_K4242012](http://parts.igem.org/Part%3ABBa_K4242012)) Transcription of the reporter gene *gfp* is promoted by cI promoter in the absence of FleQ. We found that the fluorescence of recombinant *E. coli*/pHYD-2 and *S. oneidensis*/pHYD-2 was drastically higher than that of the strains harboring pHYD-1 (Fig. 4) , indicating that the tandem promoter is capable of constitutively promoting *gfp* expression in the absence of FleQ in both strains.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/wet-pho/7.png)

Fig. 2 a|Structure of pHYD-2 b|Colony RPC result of pHYD-2

# **Cycle 3: Add the existence of FleQ**

---

To test whether FleQ can repress *gfp* expression under the control of the tandem promoter, we cloned *fleQ* gene from the genome of *P. aeruginosa* and inserted it into pHYD-2, generating the plasmid pHYD-3 ([BBa\_K4242013](http://parts.igem.org/Part%3ABBa_K4242013)) that contains the complete c-di-GMP biosensor. FleQ represses the transcription of *gfp* though inhibition of the RNA polymerase to access DNA by binding to Ppel when the level of c-di-GMP is low. At high intracellular c-di-GMP, FleQ undergoes a conformational change when it binds c-di-GMP, which relieves FleQ from *pel* promoter and leads to *gfp* gene expression under the control of the tandem promoter. As FleQ represses the expression of *gfp* by binding to the promoter Ppel, the fluorescence of *E. coli*/pHYD-3 and *S. oneidensis*/pHYD-3 expressing the repressor FleQ (Fig. 4) was significantly decreased compared to that of the strains with pHYD-2 that has no FleQ. Taken together, the results showed the tandem promoter is effective in promoting expression of the reporter gene(*gfp*), and the transcription factor FleQ is able to decrease *gfp* expression level.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/wet-pho/8.png)

Fig.3 a|Structure of pHYD-3 b| Colony RPC result of pHYD-3

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/wet-pho/9.png)

Fig.4 a|engineering vectors b| Normalized fluorescence intensity in *E.coli* BL21
c| Normalized fluorescence intensity in *S. oneidensis* MR-1

{% endblock %}


![](https://static.igem.wiki/teams/4242/wiki/background/b-res.png)

Results

{% include "sider.html" %}

[Part I](#Part I)
[Part II](#Part II)
[Part III](#Part III)
[Part IV](#Part IV)
[Part V](#Part V)
[Conclusion](#Conclusion)
[Reference](#Reference)

# Part I: Biobrick Construction and transformation

---

Work we have done

We have constructed biobrick pHYD-1 ([BBa\_K4242011](http://parts.igem.org/Part%3ABBa_K4242011)) in which reporter gene *gfp* is cloned at the downstream of promoter Ppel for testing the performance of promotor Ppel in *E. coli* and *S. oneidensis*. The key gene parts are shown in Fig. 1a. The ligation products were transferred to *E. coli*. To select colonies with pHYD-1, colony PCR was conducted, and the result is shown in Fig. 1b. Finally, the constructs were sequenced.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/1111.png)

Fig. 1 a|Structure of pHYD-1 b| Colony PCR result of pHYD-1

We have constructed a tandem promoter PcI/pel by using overlap PCR to fuse a constitutive promoter PcI with Ppel. The PCR products directly were inserted at the upstream of *gfp* with a RBS. The resultant biobrick is named as pHYD-2 ([BBa\_K4242012](http://parts.igem.org/Part%3ABBa_K4242012)) for testing the performance of tandem promotor PcI/pel in *E. coli* and *S. oneidensis*. The structure of pHYD-2 is shown in Fig 2a. The ligation products were transferred into *E. coli*. Then, we conducted colony PCR to select colonies with pHYD-1, and the result is shown in Fig. 2b. Finally, the constructs were sequenced.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/2222.png)

Fig. 2 a|Structure of pHYD-2 b|Colony RPC result of pHYD-2

To test whether the *fleQ* can repress the *gfp* transcription driven by the tandem promoter PcI/pel in *E. coli* and *S. oneidensis*, we have constructed biobrick pHYD-3 ([BBa\_K4242013](http://parts.igem.org/Part%3ABBa_K4242013)) in which the operon of *fleQ* is placed in the front of PcI/pel- *gfp*. *fleQ* was cloned from P. aeruginosa genome. The transcription of *fleQ* is promoted by a constitutive promoter PcI. Additionally, double terminators were placed after *fleQ* and reporter genes, in order to minimize the background signal caused by gene expression leakage. The key gene parts are shown in Fig 3a. The constructs were selected by colony PCR and sequenced (Fig. 3b).

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/3333.png)

Fig.3 a|Structure of pHYD-3 b| Colony PCR result of pHYD-3

To decrease intracellular c-di-GMP levels in *E. coli* and *S. oneidensis* for testing the performance of c-di-GMP biosensor, we have constructed the part Ptac-*yhjH* ([BBa\_K4242017](http://parts.igem.org/Part%3ABBa_K4242017)) based on the existing part ([BBa\_K4242001](http://parts.igem.org/Part%3ABBa_K4242001)). *yhjH* gene was cloned using *E. coli* genome as templates. The expression of *yhjH* is controlled by IPTG-inducible promoter Ptac (Fig. 4a). By adding different concentration of IPTG to control the expression level of *yhjH*, the intracellular c-di-GMP concentrations can be controlled at different levels. Fig 4b shows the results of electrophoresis gel of pSB3C5 cut by EcoRI.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/4444.png)

Fig.4 a|Structure of pSB3C5-*yhjH* b| The result of pSB3C5-*yhjH* digested by restriction enzyme EcoR I

# Part II : Demonstration of tandem promotor and **fleQ**

---

In this work, we describe the design of a transcription-based fluorescent biosensor that is readily adaptable for gauging c-di-GMP levels in different bacteria. The components of the reporter comprise a transcription factor *fleQ* that is originated from P. aeruginosa, a tandem promoter PcI/pel that is composed of a constitutive promoter PcI and a c-di-GMP responsive promoter Ppel, as well as a fluorescent protein *gfp* as a reporter. In P. aeruginosa, the transcriptional factor *fleQ* represses expression of the pel operon for Pel exopolysaccharide biosynthesis by a simple roadblock mechanism in which *fleQ* sits at the pel promoter and prevents RNA polymerase from binding, while *fleQ* derepresses gene expression due to the conformational change of *fleQ* upon binding c-di-GMP[1]. Theoretically, the gene under pel promoter can be constitutively expressed in the bacteria that have no repressor *fleQ*[2]. To test this hypothesis, we directly fused pel promoter to *gfp* and transferred the construct into two module organisms, *E. coli* BL21 and *S. oneidensis* MR-1. However, MR-1/pHYD-1 and BL21/pHYD-1 produced very low fluorescence, which suggests the promoter Ppel is non-functional in these two bacteria (Fig. 5).

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/5555.png)

Fig.5 PcI/pel constitutively promotes expression of *gfp*. *fleQ* represses expression level of gene under the control of PcI/pel

To address this challenge, we designed a tandem promoter PcI/pel in which the transcription start site of the constitutive promoter PcI is immediately followed by the *fleQ* binding site of the promoter Ppel. Transcription of the reporter gene *gfp* is promoted by cI promoter in the absence of *fleQ*. As shown in Fig. 5, the result shows that MR-1/pHYD-2 and BL21/pHYD-2 produced high fluorescence, indicting the tandem promotor is able to trigger the reporter gene expression. Then we cloned the regulator *fleQ* into the system. The *fleQ* successfully binds to the box and reduced the *gfp* expression in MR-1/pHYD-3 and BL21/pHYD-3 as we expected, indicating the whole system is functional in chassis like *E. coli* and *S. oneidensis* (Fig. 5). We summarized the construction process and results in Fig. 6. For more information and details about this engineering cycles, please check <https://2022.igem.wiki/cug-china/engineering>

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/6666.png)

Fig.6 a| Constructed vectors b| Fluorescence intensity in *E. coli* BL21
c| Fluorescence intensity in *S. oneidensis* MR-1

# Part III : Demonstration of c-di-GMP responding to c-di-GMP

---

The native c-di-GMP can bind with *fleQ*, making biosensor produce a background fluorescence. When c-di-GMP decreases, *fleQ* further represses the *gfp* expression to decrease fluorescent outputs. To demonstrate that our biosensor can respond to different intracellular c-di-GMP concentration and indicate its decrease, we decided to achieve the decrease gradient of intracellular c-di-GMP. To achieve it, we improved an existing part ([BBa\_K4242001](http://parts.igem.org/Part%3ABBa_K4242001)) encoding the *yhjH* protein, a c-di-GMP hydrolase. By changing the promotor from T7 to Ptac, we can use IPTG to control the expression level of YjhH, reducing c-di-GMP concentration at different level (Fig. 7). We transformed this biobrick into the BL21/pHYD-3 and MR-1/pHYD-3, obtaining the strains BL21/ (pHYD-3+pSB3C5-*yhjH*) and MR-1/(pHYD-3+pSB3C5-*yhjH*), as well as their responding control strain BL21/(pHYD-3+pSB3C5-Ptac) and MR-1/(pHYD-3+pSB3C5-Ptac) (Fig. 7).

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/7777.png)

Fig.7 Structure of the pSB3C5-Ptac and pSB3C5-*yhjH*

We cultured *E. coli* with the addition of different concentrations of IPTG. Compared with the control strains, we observed a significant decrease in fluorescent signals when the IPTG concentration is above 0.01 mM after 12 hours (Fig. 8). This result shows that, the fluorescence intensity of strains with pHYD-3 and pSB3C5-*yhjH* decreased with the increase in the concentrations of IPTG, whereas the fluorescence intensities of the control strains, i.e., BL21/(pHYD-3+pSB3C5-Ptac), had no change with the increase in IPTG concentration.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/8888.png)

Fig.8 Fluorescence in BL21/pHYD-3 in different c-di-GMP levels.

We used MR-1/(pHYD-3+pSB3C5-YjhH) and MR-1/(pHYD-3+pSB3C5-Ptac) to conduct similar experiments for testing the performance of c-di-GMP biosensor in MR-1. As shown in Fig. 9, there was no difference between the fluorescent signal of cultures without IPTG and that with 0.01 mM IPTG. However, compared to them, the fluorescent signals were significantly reduced when the concentrations of IPTG is above 0.1 mM. Meanwhile, the fluorescent signals of MR-1/(pHYD-3+pSB3C5-Ptac) had no significant difference. Collectively, our results showed that IPTG had no impact on the production of fluorescent proteins, and our biosensor can convert the decrease in c-di-GMP levels to the decreased fluorescence which is easily detected.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/9999.png)

Fig.9 Fluorescence intensity of MR-1/pHYD-3 with different c-di-GMP levels.

# Part IV : Verification of biosensor screening biofilm dispersal agents

---

A group of structurally diverse compounds are known to hinder the formation of bacterial biofilm or trigger the dispersal of biofilm.[3] It is also known that biofilm dispersal can be induced by sequestering cellular c-di-GMP, which indicates that a low c-di-GMP level could directly lead to biofilm dispersal. By using the biosensor, we thought whether some of the biofilm-dispersing compounds that cause a reduction in cellular c-di-GMP levels can be selected in *E. coli* and *S. oneidensis*. The answer to the question would determine whether we can apply our biosensor into the development of new chemicals or technical to repress the biofilm formation. Based on the reported study, the compounds including D-Tyrosine, 3-Iodolylacetonitrile and Resveratrol are used in our projects (Fig. 10). The concentrations of the biofilm-dispersing compounds used in this project are listed in Table S1[3] in the page ([https://2022.igem.wiki/cug-china/proof of concept](https://2022.igem.wiki/cug-china/proof%20of%20concept)). It was reported that these compounds are able to disperse biofilm in *E. coli* and other bacterial strains. The measurement of the fluorescence intensity were performed following the protocol in the protocol page<https://2022.igem.wiki/cug-china/protocol>

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/0000.png)

Fig.10 Biofilm-dispersing compounds used in our experiments.

The strains BL21/pHYD-3 and MR-1/pHYD-3 were treated with biofilm-dispersing agents mentioned above. The individual compounds were added separately to the medium at the beginning of the cell culture. Then they were incubated at 37℃ and 30℃ for 12 hours, respectively. Then we harvested the cells and measured fluorescence by the protocol. As shown in Fig. 11, a decrease of the fluorescence intensity was observed for all three compounds in BL21 strains. Compared with the control group, i.e., BL21/pHYD-3 without the treatment of biofilm-dispersing agents, these three compounds respectively reduced fluorescence intensity about 23%, 27% and 13%. The results are consistent with the reported study.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/01010.png)

Fig.11 Fluorescence intensity of MR-1/pHYD-3, cultured with different dispersal agents. Two-sided Student's t test was used to analyze the statistical significance (\*P< 0.05)

Similarly, we treated the strain MR-1/pHYD-3 with these biofilm-dispersing agents. Compared to the control group in which MR-1/pHYD-3 cells were grown without biofilm-dispersing agents, the treatments of biofilm-dispersing agents significantly reduced the fluorescence of MR-1/pHYD-3 that containing our c-di-GMP biosensor (Fig. 12). The results suggested that these compounds could reduce intracellular c-di-GMP level of MR-1.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/1313.png)

Fig.12 Fluorescence intensity of MR-1/pHYD-3, cultured with different dispersal agents. Two-sided Student's t test was used to analyze the statistical significance (\*P< 0.05)

To verify whether these compounds indeed reduced the intracellular c-di-GMP of MR-1, we cultured the strains again under the same condition and harvest the cells to measure its intracellular c-di-GMP concentration. On the basis of the curves measured by HPLC, we observed that the compounds caused a drop of intracellular c-di-GMP concentration from 1.71 pmol/μg protein to 0.44 pmol/μg total protein (Fig. 13), which was reduced by about 73%. These results suggested that our biosensor is able to monitor these compounds inducing biofilm dispersal by reducing intracellular c-di-GMP level.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/1414.png)

Fig.13 C-di-GMP level in MR-1/pHYD-3 measured by HPLC.

The biosensors revealed a decline of c-di-GMP level when the cells were treated with biofilm-dispersing agents. The result establishes the biosensor as valuable tool for using in chemical biology and biofilm-dispersal agent screening. However, analyzing the data of our experiments also exhibit some problems in our biosensor. As shown in Fig.11, 12 and 13, when treated with biofilm dispersal agents for 6 hours, the reduction of fluorescence varies between 10%-27%, which is out of proportion with the decrease of c-di-GMP level (about 73%). It means the dynamic range of the detection is not wide enough. Although it can indicate the drop of c-di-GMP level, it can't precisely reflect the dynamic change of c-di-GMP due to its narrow dynamic range,

# Part V Work has not finished yet

---

Based on our current data, we found that our biosensor exhibits a narrow dynamic range. One of factor for the narrow dynamic range could because of the low native c-di-GMP level or the high expression level of *fleQ*. We cannot change the native c-di-GMP level, but can control the expression level of *fleQ* by using inducible promoter. Thus, we will preliminary improve the dynamic range of our biosensor by controlling the expression of regulator *fleQ* with a arabinose inducible promoter PBAD. The biobrick design is shown in Fig. 14a. The constitutive promoter PcI in pHYD-3 is replaced by the inducible promoter PBAD, and the resultant biobrick is named pHYD-B1.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/npcsb.png)

Fig.14 a|improvement of pHYD-3 by changing promotor. b|Colony RPC result of pHYD-B1

It has been reported that *fleQ* can act as the activator of Ppel in the presence of another regulator *fleN*. To further widen the dynamic range of this c-di-GMP biosensor, we cloned regulator gene *fleN* into pHYD-B1, resulting in the biobrick pHYD-B2 (Fig. 15). Although we have constructed these biobricks ([BBa\_K4242014](http://parts.igem.org/Part%3ABBa_K4242014), [BBa\_K4242015](http://parts.igem.org/Part%3ABBa_K4242015)) and added these parts in the registry, their demonstration have note done because of the limited time.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/jzl.png)

Fig.15 a|improvement of pHYD-B1 by adding another regulator *fleN*. b|Colony RPC result of pHYD-B2

We also inserted pHYD-B1 into the *E. coli* BL21 and measured its growth curves under different arabinose concentrations (Fig. 16). However, we don't have enough time to finish further experiments for their demonstration. The results showed that the increase in arabinose concentrations slightly reduced the growth rate of *E. coli* BL21.

![Pulpit rock](https://static.igem.wiki/teams/4242/wiki/results/zfx.png)

Fig.16 Growth curves of *E. coli* BL21/pHYD-B1 under different Arabinose concentrations.

We have constructed the gene part pSB3C5-*yhjH* to decrease intracellular c-di-GMP concentration at different levels for testing the performance of c-di-GMP biosensor, simulating the effect of biofilm-dispersing agents that can induce biofilm dispersal by decreasing c-di-GMP levels. In addition to “bad” biofilms, there are a lot of “good” biofilms which have a wide range of biotechnology applications. For example, *S. oneidensis* is a model electroactive bacterium that can exchange electron with electrodes in bioelectrochemical systems (BESs). Specially, it shows promise as biocatalysts for efficient conversion of a wide range of organic wastes and renewable biomass to electricity. This application is greatly associated with the formation of *S. oneidensis* biofilms on the anode. Increasing c-di-GMP level to enhance anode biofilms of *S. oneidensis* is a promising strategy to promote electron transfer and increase electricity generation. Our biosensors can be used to screening the c-di-GMP synthase with high enzyme activity. Therefore, we need to check whether our biosensor can detect the increase in c-di-GMP levels. To increase intracellular c-di-GMP level, we cloned a c-di-GMP synthase gene, yedQ. We have finished the construction of this part ([BBa\_K4242016](http://parts.igem.org/Part%3ABBa_K4242016)) and plan to finish its demonstration in next year.

# Conclusion

---

Collectively, we can see a clear path of the engineering cycles and construction of the biosensor, testing its reaction to both endogeneity and exogenous decrease of c-di-GMP level. The biosensor also revealed a decline of c-di-GMP level wen the cells were treated with biofilm dispersing agents. Now we can come to conclusion that we have successfully developed a broadly applicable biosensor for gauging intracellular c-di-GMP levels, which can be used to screening biofilm dispersal agents and other biofilm/c-di-GMP associated research. By studying the biosensor-containing cells embedded in biofilm matrixes, we will also be able to gain a better understanding of the roles c-di-GMP played in the highly dynamic and complex processes of biofilm formation and dispersal. In future, we will continue to widen the dynamic range of our biosensor and explore its wider applications, which matches the goal of applying synthetic biology to make the world better.

# Reference

---

[1] Baraquet C, Murakami K, Parsek M R, et al. The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP[J]. Nucleic Acids Res, 2012,40(15):7207-7218.

[2] Hickman J W, Harwood C S. Identification of FleQ from Pseudomonas aeruginosa as ac-di-GMP-responsive transcription factor[J]. Molecular microbiology, 2008,69(2):376-389.

[3] Ho C L, Chong K S J, Oppong J A, et al. Visualizing the perturbation of cellular cyclic di-GMP levels in bacterial cells[J]. Journal of the American Chemical Society, 2013,135(2):566-569.