iGEM 2019 — Team Hong_Kong-CUHK

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# Team:Hong Kong-CUHK/Notebook

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[HOME](https://2019.igem.org/Team%3AHong_Kong-CUHK)

PROJECT

[Description](https://2019.igem.org/Team%3AHong_Kong-CUHK/Description)
[Design](https://2019.igem.org/Team%3AHong_Kong-CUHK/Design)
[Demonstrate](https://2019.igem.org/Team%3AHong_Kong-CUHK/Demonstrate)
[Improve](https://2019.igem.org/Team%3AHong_Kong-CUHK/Improve)
[Attributions](https://2019.igem.org/Team%3AHong_Kong-CUHK/Attributions)
[Safety](https://2019.igem.org/Team%3AHong_Kong-CUHK/Safety)

WET LAB

[Experiments](https://2019.igem.org/Team%3AHong_Kong-CUHK/Experiments)
[Results](https://2019.igem.org/Team%3AHong_Kong-CUHK/Results)
[Notebook](https://2019.igem.org/Team%3AHong_Kong-CUHK/Notebook)
[Contribution](https://2019.igem.org/Team%3AHong_Kong-CUHK/Contribution)

DRY LAB

[Homology Study](https://2019.igem.org/Team%3AHong_Kong-CUHK/Homology_Study)
[Testification](https://2019.igem.org/Team%3AHong_Kong-CUHK/Testification)
[Promoter Activity](https://2019.igem.org/Team%3AHong_Kong-CUHK/Promoter_Activity)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3AHong_Kong-CUHK/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AHong_Kong-CUHK/Public_Engagement)

TEAM

[Team Members](https://2019.igem.org/Team%3AHong_Kong-CUHK/Team)
[Collaborations](https://2019.igem.org/Team%3AHong_Kong-CUHK/Collaborations)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AHong_Kong-CUHK/Parts)
[Basic Parts](https://2019.igem.org/Team%3AHong_Kong-CUHK/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AHong_Kong-CUHK/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AHong_Kong-CUHK/Part_Collection)

AWARDS

[Award summary

[Measurement](https://2019.igem.org/Team%3AHong_Kong-CUHK/Measurement)
[Model](https://2019.igem.org/Team%3AHong_Kong-CUHK/Model)](https://2019.igem.org/Team%3AHong_Kong-CUHK/Award%20Summary)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Hong_Kong-CUHK)

# Notebook

*done by wet lab team member Heman, Savio, Billy, Greenie and Joanna*

##### **May 7 to May 15 (literature review):**

1. Reviewing on sequence data of *Xcm* RpfC and RpfG

2. Designing primer and restriction site for the afterwards cloning

3. Designing genes fragments with restriction sites on both ends

##### **May 16 to May 29 (literature review):**

1. Codon optimization for genes on online database

##### **June 3 to June 14 (stage 1 cloning):**

1. Competent cell preparation

##### **June 17 (stage 1 cloning):**

1. PCR of synthesised gene fragments(RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed)

2. pRSFDuet-1 extraction from E. coli clone

##### **June 18 (stage 1 cloning):**

1. RE digestion(EcoRI, PstI) of insert (RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed) and vectors (pRSFDuet-1)

2. Ligation of genes to vector

3. Transformation of bacterial cloning strain, DH-5α

##### **June 19 to 20 (stage 1 cloning):**

1. Picking clones from transfromed bacteria

2. Culturing overnight

3. Collecting plasmid with clones from overnight culture

4. RE mapping to confirm extistence of cloned genes

##### **June 21 to July 3 (stage 1 cloning):**

1. Repeating procedures, starting from PCR, with the newly prepared competent cell, new sets of primers and optimised annealing temperature as no positive clone could be obtain after transformation. It was suspected that problem occurs mainly in the design of primers.

2. Collecting plasmid with gene inserts, RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed (stage 1 clones)

##### **July 8 to July 12 (stage 2 and 3 cloning):**

1. RE diegestion of inserts (RpfC, RpfG, Clp-TAD, Sigma 54, CBP, eforRed) by XbaI and SpeI

2. Ligation of RpfC-RpfG (A), Clp-Sigma 54 (B) and CBP-eforRed (C)

3. Ligation of A, B and C into 3 separate vectors of pRSFDuet

4. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21

5. Picking clones from transfromed bacteria

6. Culturing overnight

7. Collecting plasmid with clones from overnight culture (stage 2 clones)

8. RE mapping to confirm extistence of cloned genes

##### **July 15 to July 26 (stage 2 and 3 cloning):**

1. PCR of construct A, B and C.

2. Adding the EcoRI, PstI restriction sites to construct A and the NdeI, XhoI restriction sites to contruct B by the primer pairs

3. RE diegestion of construct A, B and C, and the empty vector pRSFDuet-1

4. Ligation of A, B and C into 3 separate vectors of pRSFDuet, according to their respective new RE sites

5. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21

6. Picking clones from transfromed bacteria

7. Culturing overnight

8. Collecting plasmid with clones from overnight culture (stage 3 clones)

9. RE mapping to confirm extistence of cloned genes

##### **July 29 to August 9 (stage 4 & 5 cloning):**

1. RE diegestion of construct A and the vector with construct C

2. Ligation of A into vector with construct C

3. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21

4. Picking clones from transfromed bacteria

5. Culturing overnight

6. Collecting plasmid with clones from overnight culture (stage 4 clones)

7. RE mapping to confirm extistence of cloned genes

##### **July 12 to August 16 (stage 4 & 5 cloning):**

1. RE diegestion of construct B and the vector with construct A and C

2. Ligation of B into vector with construct A and C

3. Transformation of bacterial cloning strain, DH-5α, and expression strain, BL-21

4. Picking clones from transfromed bacteria

5. Culturing overnight

6. Collecting plasmid with clones from overnight culture (stage 5 clones)

7. RE mapping to confirm extistence of cloned genes

##### **August 28 to August 30 (stage 4 & 5 cloning):**

1. Transform final construct into BL21 competent cell

2. Starter culture and IPTG induction (1mM)

3. Collect time points 12hr, 16hr, 20hr, 24hr, 28hr

##### **September 1 to October 4 (assays):**

1. protein expression of stage 1 and 5 clones

- western blots of construct A, B and C separately

2. optimization confirmation

- western blot of RpfG protein, comparing the previous iGEM team biobrick and the RpfG biobrick optimized by our team

3. eforRed expression and signal generation

- qPCR of eforRed transcripts under 1 mM IPTG induction from the time interval of 0 to 18 hour, detecting the time-dependent transcription level

- optical density of eforRed-expressing stage 5 clones after 0-10 mM IPTG treatment, ranging from 1 to 8 hours induction time

4. DSF treatment assay

- qPCR of eforRed transcripts under 0 to 1 mM DSF treatment for 16 hour, detecting the dosage-dependent response of engineered E. Coli to DSF stimulation

5. cyclic-di-GMP suppressive drug assay

- qPCR of eforRed transcripts under 25 to 800 ug/mL azathioprine and sulfathiazole separate treatment for 16 hour, confirming the expression activation due to cyclic-di-GMP suppression


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# Team:Hong Kong-CUHK/Experiments

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PROJECT

[Description](https://2019.igem.org/Team%3AHong_Kong-CUHK/Description)
[Design](https://2019.igem.org/Team%3AHong_Kong-CUHK/Design)
[Demonstrate](https://2019.igem.org/Team%3AHong_Kong-CUHK/Demonstrate)
[Improve](https://2019.igem.org/Team%3AHong_Kong-CUHK/Improve)
[Attributions](https://2019.igem.org/Team%3AHong_Kong-CUHK/Attributions)
[Safety](https://2019.igem.org/Team%3AHong_Kong-CUHK/Safety)

WET LAB

[Experiments](https://2019.igem.org/Team%3AHong_Kong-CUHK/Experiments)
[Results](https://2019.igem.org/Team%3AHong_Kong-CUHK/Results)
[Notebook](https://2019.igem.org/Team%3AHong_Kong-CUHK/Notebook)
[Contribution](https://2019.igem.org/Team%3AHong_Kong-CUHK/Contribution)

DRY LAB

[Homology Study](https://2019.igem.org/Team%3AHong_Kong-CUHK/Homology_Study)
[Testification](https://2019.igem.org/Team%3AHong_Kong-CUHK/Testification)
[Promoter Activity](https://2019.igem.org/Team%3AHong_Kong-CUHK/Promoter_Activity)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3AHong_Kong-CUHK/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AHong_Kong-CUHK/Public_Engagement)

TEAM

[Team Members](https://2019.igem.org/Team%3AHong_Kong-CUHK/Team)
[Collaborations](https://2019.igem.org/Team%3AHong_Kong-CUHK/Collaborations)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AHong_Kong-CUHK/Parts)
[Basic Parts](https://2019.igem.org/Team%3AHong_Kong-CUHK/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AHong_Kong-CUHK/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AHong_Kong-CUHK/Part_Collection)

AWARDS

[Award summary

[Measurement](https://2019.igem.org/Team%3AHong_Kong-CUHK/Measurement)
[Model](https://2019.igem.org/Team%3AHong_Kong-CUHK/Model)](https://2019.igem.org/Team%3AHong_Kong-CUHK/Award%20Summary)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Hong_Kong-CUHK)

# Experiments

![](https://static.igem.org/mediawiki/2019/2/24/T--Hong_Kong-CUHK--experiment_flow.png)

The whole project in general included 3 parts, the literature review, cloning and assays. The assays were further divided into 5 specific goal, which were the confirmation of protein expression in the produced clones, the measurement of codon optimization effectiveness, testify the generation of eforRed signal and the drug and DSF treatment assays.

Literature review could help us in the planning of the whole project. Details like the protein tag for purification, cell culturing, gene expression condition, gene sequence variation in different bacterial strains were the main objectives in this step. Besides, codon optimization was also included in this planning process. As we knew that different organism strains would have different preferences in codon translation, we did codon optimization in the online database system in order to design the gene sequences which were the most suitable to be expressed in E. Coli.

![](https://static.igem.org/mediawiki/2019/4/4d/T--Hong_Kong-CUHK--biobrick_stage.png)

Figure 1: Cloning stages of the project. Stage 1 involves 6 separate inserts. Stage 2 and 3 involves pair of inserts in each plasmid, named as the construct A, B and C. Stage 4 and 5 involves the ligation of the 3 constructs to the same plasmid.

![](https://static.igem.org/mediawiki/2019/9/92/T--Hong_Kong-CUHK--biobrick_scar.png)

Figure 2: Stage 2 involves the irreversible ligation of insert pairs by the restriction site XbaI and SpeI. As XbaI and SpeI have the same sticky end, they can be ligated with each other. Once ligation is done, the site cannot be neither recognised by XbaI enzyme nor SpeI enzyme.

The next step in our project was the cloning (figure 1). The biobrick cloning involved 5 stages as shown in the figure. Overall, we had to insert 6 biobricks into a plasmid for transformation, which was a difficult task to attain. To made sure the cloning progress could be monitored, we divided the biobricks to be cloned in pairs, which were called the “constructs” in our project description. A biobrick “construct” contained two gene inserts that were ligated by the XbaI and SpeI restriction sites, forming a scar which could not be digested anymore (figure 2). The formation of scar ensured that unnecessary restriction digestion would not happen in the further cloning stages.

a)

![](https://static.igem.org/mediawiki/2019/0/02/T--Hong_Kong-CUHK--protein_expression.png)

b)

![](https://static.igem.org/mediawiki/2019/4/4a/T--Hong_Kong-CUHK--eforRed_signal_generation_assay.png)

c)

![](https://static.igem.org/mediawiki/2019/c/cd/T--Hong_Kong-CUHK--DSF_assay.png)

Figure 3: (a-c) The figures showed assays in th project.

The last step would be the assay part (figure 3). We would like to first ensure proteins involving in the intracellular signal transduction can be expressed. Therefore we did the western blots of RpfC, RpfG and Clp proteins. Next, testing the expression efficiency of codon-optimized RpfG was important to prove the success in our work. In this assay, RpfG of old sequence and of the optimized sequence were expressed in E. Coli. After confirming the signal transduction part expression, the next assay target would be the signaling protein, eforRed. Drug assays were to prove that whether the molecular pathway of this system works. The drugs used were azathioprine and sulfathiazole. Both of them were the c-di-GMP suppressive drug. In our project idea, we expected that after suppressing the c-di-GMP level, Clp protein would be free to bind DNA and promote gene transcription. The assay helped us to test the relationship between the c-di-GMP level, Clp DNA-binding affinity, and the eforRed expression. Last but not least, DSF assay was the final goal of the molecular aspect of the system. We aimed to modify the E. Coli and make it be able to detect and respond to DSF molecules in the culture environment. In this DSF assay, cells were treated with DSF in different concentration and were tested for the eforRed expression.

## Lab manual

<[Lab manual: Agarose gel preparation](https://static.igem.org/mediawiki/2019/c/c0/T--Hong_Kong-CUHK--manual_agar_gel.pdf)>

<[Lab manual: buffer list](https://static.igem.org/mediawiki/2019/d/d1/T--Hong_Kong-CUHK--manual_buffer_list.pdf)>

<[Lab manual: cell culture](https://static.igem.org/mediawiki/2019/4/41/T--Hong_Kong-CUHK--manual_cell_culture.pdf)>

<[Lab manual: competent cell preparation](https://static.igem.org/mediawiki/2019/b/bf/T--Hong_Kong-CUHK--manual_com_cell.pdf)>

<[Lab manual: DSF treatment for qPCR](https://static.igem.org/mediawiki/2019/0/03/T--Hong_Kong-CUHK--manual_DSF_qPCR.pdf)>

<[Lab manual: IPTG induction for OD measurement](https://static.igem.org/mediawiki/2019/2/22/T--Hong_Kong-CUHK--manual_IPTG_OD.pdf)>

<[Lab manual: IPTG induction for qPCR](https://static.igem.org/mediawiki/2019/b/bd/T--Hong_Kong-CUHK--IPTG_qPCR.pdf)>

<[Lab manual: PCR](https://static.igem.org/mediawiki/2019/f/f8/T--Hong_Kong-CUHK--manual_PCR.pdf)>

<[Lab manual: restriction digestion](https://static.igem.org/mediawiki/2019/c/c1/T--Hong_Kong-CUHK--manual_RE.pdf)>

<[Lab manual: SDS PAGE and western blot](https://static.igem.org/mediawiki/2019/3/3f/T--Hong_Kong-CUHK--manual_SDS_PAGE_WB.pdf)>

<[Lab manual: sonication](https://static.igem.org/mediawiki/2019/9/94/T--Hong_Kong-CUHK--manual_sonication.pdf)>

<[Lab manual: suppressive drug treatment](https://static.igem.org/mediawiki/2019/c/c3/T--Hong_Kong-CUHK--manual_suppressive_drug_assay.pdf)>

<[Lab manual: ligation and transformation](https://static.igem.org/mediawiki/2019/0/09/T--Hong_Kong-CUHK--manual_ligation_and_transform.pdf) >

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# Team:Hong Kong-CUHK/Results

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[HOME](https://2019.igem.org/Team%3AHong_Kong-CUHK)

PROJECT

[Description](https://2019.igem.org/Team%3AHong_Kong-CUHK/Description)
[Design](https://2019.igem.org/Team%3AHong_Kong-CUHK/Design)
[Demonstrate](https://2019.igem.org/Team%3AHong_Kong-CUHK/Demonstrate)
[Improve](https://2019.igem.org/Team%3AHong_Kong-CUHK/Improve)
[Attributions](https://2019.igem.org/Team%3AHong_Kong-CUHK/Attributions)
[Safety](https://2019.igem.org/Team%3AHong_Kong-CUHK/Safety)

WET LAB

[Experiments](https://2019.igem.org/Team%3AHong_Kong-CUHK/Experiments)
[Results](https://2019.igem.org/Team%3AHong_Kong-CUHK/Results)
[Notebook](https://2019.igem.org/Team%3AHong_Kong-CUHK/Notebook)
[Contribution](https://2019.igem.org/Team%3AHong_Kong-CUHK/Contribution)

DRY LAB

[Homology Study](https://2019.igem.org/Team%3AHong_Kong-CUHK/Homology_Study)
[Testification](https://2019.igem.org/Team%3AHong_Kong-CUHK/Testification)
[Promoter Activity](https://2019.igem.org/Team%3AHong_Kong-CUHK/Promoter_Activity)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3AHong_Kong-CUHK/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AHong_Kong-CUHK/Public_Engagement)

TEAM

[Team Members](https://2019.igem.org/Team%3AHong_Kong-CUHK/Team)
[Collaborations](https://2019.igem.org/Team%3AHong_Kong-CUHK/Collaborations)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AHong_Kong-CUHK/Parts)
[Basic Parts](https://2019.igem.org/Team%3AHong_Kong-CUHK/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AHong_Kong-CUHK/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AHong_Kong-CUHK/Part_Collection)

AWARDS

[Award summary

[Measurement](https://2019.igem.org/Team%3AHong_Kong-CUHK/Measurement)
[Model](https://2019.igem.org/Team%3AHong_Kong-CUHK/Model)](https://2019.igem.org/Team%3AHong_Kong-CUHK/Award%20Summary)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Hong_Kong-CUHK)

Lab

# Wet Lab-Cloning

### Stage 1

In stage 1, vector used is the pRSF-Duet1 from own storage clone and inserts included are the RpfC, RpfG, Clp-PspF, sigma 54, CBS, eforRed, synthesised by the INTEGRATED DNA TECHNOLOGIES (IDT). Protocols used are on the protocols page. With the aid of PCR, the fragments was amplified for further cloning processes. The optimal conditions, which is enclosed in the protocol page, were found after numerous tests.

![](https://static.igem.org/mediawiki/2019/f/ff/T--Hong_Kong-CUHK--stage_1_pic1a.png)
![](https://static.igem.org/mediawiki/2019/d/d2/T--Hong_Kong-CUHK--stage_1_pic1b.png)
![](https://static.igem.org/mediawiki/2019/9/90/T--Hong_Kong-CUHK--stage_1_pic_1c.png)

Figure 1: 1% agarose gel showing the results of PCR. Right) the bands of RpfC and RpfG. Middle) the bands of Clp-PspF and sigma 54. Left) the bands of CBS and eforRed.

Then we performed restriction digest for afterward insert ligation. To prevent self-ligation, double digestion and alkaline phosphatase were used. The ligated plasmids were then successfully transformed into E. coli.

![](https://static.igem.org/mediawiki/2019/9/92/T--Hong_Kong-CUHK--stage_1_RE_2.jpeg)

Figure 2: 1% agarose gel showing the results of restriction digestion. Lanes 3 to 4 are the uncut control and single cut control, showing the viability of enzyme used. Lanes 6 to 8 are the double cut vector, pRSFduet. Lanes 10 to 15 are the double-cut inserts.

After transformation, colonies were picked and colony PCR were performed to verify the existence of inserts in clones.

![](https://static.igem.org/mediawiki/2019/3/36/T--Hong_Kong-CUHK--culture_PCR.jpeg)

Figure 3: 1% agarose gel showing the results of colony PCR. 3 colonies were picked for each insert. At least one successful were obtained for each insert. Correct band sizes and locations were labelled. The faint bands at the bottom were suspected to be the primer dimers. Regarding the lanes of CBS, the CBS bands were overlapped with the primer dimers.

### Stage 3

In stage 3, PCR was carried out to change the restriction sites of the constructs. Construct A, the RpfC-RpfG insert, would contain the EcoRI and PstI sites. Construct B, the Clp\_pspF\_TAD-sigma 54 insert, would contain NdeI and XhoI sites. Construct C, CBS-eforRed insert, would contain XbaI and SpeI sites. After PCR, restriction digestion, ligation and transformation were followed. The existence of positive clones was confirmed by colony PCR (figure 4).

![](https://static.igem.org/mediawiki/2019/a/a1/T--Hong_Kong-CUHK--construct_A.jpeg)

Figure 4a: 1% agarose gel; Clones containing construct A were obtained. Clone 1, 2, 3, 5, 6, 8, 10, 11, 12, 13, 15, 16 showed positive results.

![](https://static.igem.org/mediawiki/2019/d/d6/T--Hong_Kong-CUHK--construct_B.jpeg)

Figure 4b: 1% agarose gel; Clones containing construct B were obtained. All clones picked showed positive results.

![](https://static.igem.org/mediawiki/2019/4/43/T--Hong_Kong-CUHK--construct_C.jpeg)

Figure 4c: 1% agarose gel; Clones containing construct C were obtained. Clone 1, 2, 3, 4 showed positive results.

### Stage 5

In stage 5, construct A, B and C were ligated in series in the same plasmid, forming the final cloning product. (figure 5)

![](https://static.igem.org/mediawiki/2019/7/71/T--Hong_Kong-CUHK--clone_AB%2BC.jpeg)

Figure 5: 1% agarose gel showing the result of colony PCR. Clones containing construct ABC were obtained. Clones from 1 to 16, except 8, showed positive results.

### Protein expression

After stages of cloning, western blotting was performed using the iBind system from Life Technologies to see if the inserts can be successfully expressed after being induced. Clones were induced by 1mM IPTG at 32oC and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr.

Clones were induced by 1mM IPTG at 32℃ and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, Clp-PspF, RpfG, RpfC proteins were confirmed with successful expression at all time points. Results of blots probing Clp (figure 6a) and RpfG (figure 6b) proteins showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells. For the blot probing RpfC (figure 6c) protein, clones collected at 16hr and 20hr showed highest target protein quantity instead.

![](https://static.igem.org/mediawiki/2019/d/d0/T--Hong_Kong-CUHK--PspF-Clp_WB.jpeg)

Figure 6a: Western blot analysis of Clp protein expression using Myc-Tag (9B11) Mouse mAb (1:2000).

![](https://static.igem.org/mediawiki/2019/2/28/T--Hong_Kong-CUHK--RpfG-FLAG_WB.jpeg)

Figure 6b: Western Blot analysis of RpfG protein expression using Sigma F7425 ANTI-FLAG® antibody (1:5000).

![](https://static.igem.org/mediawiki/2019/7/75/T--Hong_Kong-CUHK--RpfC-HA_WB.jpeg)

Figure 6c: Western Blot analysis of RpfC protein expression using Sigma Aldrich Monoclonal Anti-HA antibody(H9658) (1:20000).

### Codon optimization confirmation

![](https://static.igem.org/mediawiki/2019/d/d2/T--Hong_Kong-CUHK--codon_optimization_RpfG.jpeg)

Figure 7: Nitrocellulose membrane stained by Pierce™ Reversible Protein Stain Kit after protein transfer from SDS-PAGE gel.

Following confirmation of protein expression, codon optimization of RpfG was performed. RpfG overexpression can be clearly seen in clones after codon optimization. The amount of RpfG proteins significantly increased compared to that in the old clones. The amount also rose with a longer incubation time of IPTG. Comparing Figure 1b and Figure 2, we can deduce that RpfG expression reached saturation at 12hr and started degradation afterward.

### eforRed signal generation - Dosage and time dependent

Figure 8a: ![](https://static.igem.org/mediawiki/2019/9/95/T--Hong_Kong-CUHK--pellet_0mM_IPTG.jpeg)

Figure 8b: ![](https://static.igem.org/mediawiki/2019/3/39/T--Hong_Kong-CUHK--pellet_100nM_IPTG2.jpeg)

Figure 8c: ![](https://static.igem.org/mediawiki/2019/c/cf/T--Hong_Kong-CUHK--pellet_200nM_IPTG.jpeg)

Figure 8d: ![](https://static.igem.org/mediawiki/2019/9/98/T--Hong_Kong-CUHK--pellet_500nM_IPTG.jpeg)

Figure 8e: ![](https://static.igem.org/mediawiki/2019/d/da/T--Hong_Kong-CUHK--pellet_1mM_IPTG.jpeg)

Figure 8f: ![](https://static.igem.org/mediawiki/2019/3/34/T--Hong_Kong-CUHK--pellet_2mM_IPTG.jpeg)

Figure 8g: ![](https://static.igem.org/mediawiki/2019/5/57/T--Hong_Kong-CUHK--pellet_5mM_IPTG.jpeg)

Figure 8h: ![](https://static.igem.org/mediawiki/2019/4/43/T--Hong_Kong-CUHK--pellet_10mM_IPTG.jpeg)

Figure 8a -8h shows cells that were induced by different concentrations of IPTG, ranging from 0 mM to 10 mM. In general, red-colored pellets could be shown after 2 hours incubation with IPTG.

### eforRed signal generation - Time dependent

![](https://static.igem.org/mediawiki/2019/f/f8/T--Hong_Kong-CUHK--eforRed_signal_generation_qPCR.jpeg)

Figure 9: eforRed expression level in response to 1mM IPTG induction from the induction time of 0 to 18 hour. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). Two star signs (\*\*) indicate significance at p < 0.01. Three star signs (\*\*\*) indicate significance at p < 0.001. Four star signs (\*\*\*\*) indicate significance at p < 0.0001.

Expression eforRed was significantly increased, starting from 6 hours after induction, while it reached maximum at 10 hours after induction and started decline.

### c-di-GMP suppressive drug assay

![](https://static.igem.org/mediawiki/2019/b/b6/T--Hong_Kong-CUHK--sulfathiazole_assay2.jpeg)

Figure 10: The graph showed the fold change in eforRed expression after various concentration of sulfathiazole treatment. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). Two star signs (\*\*) indicate significance at p < 0.01. Three star signs (\*\*\*) indicate significance at p < 0.001. Four star signs (\*\*\*\*) indicate significance at p < 0.0001.

The cells were first induced for 4 hours by 1mM IPTG. Then, it was followed by the 16-hour treatment by 0-800g/mL of sulfathiazole. The cells reached the maximum increase in eforRed expression at 25ug/mL drug treatment, followed by decrease in expression level at higher drug concentrations. Results showed a significant increase in all treatment groups. The decrease in expression might be due to the cytotoxic activity of sulfathiazole.

![](https://static.igem.org/mediawiki/2019/7/7b/T--Hong_Kong-CUHK--azathioprine_assay.jpeg)

Figure 11: The graph showed the fold change in eforRed expression after various concentration of azathioprine treatment. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). Two star signs (\*\*) indicate significance at p < 0.01. Four star signs (\*\*\*\*) indicate significance at p < 0.0001.

The cells were first induced for 4 hours by 1mM IPTG. Then, it was followed by the 16-hour treatment by 0-400g/mL of azathioprine. The cells reached the maximum increase in eforRed expression at 100ug/mL drug treatment, followed by decrease in expression level at higher drug concentrations. Results showed significant increase in all treatment groups. The decrease in expression might be due to the cytotoxic activity of azathioprine.

Azathioprine and Sulfathiazole are indirect suppressor to cyclic-di-GMP, treatment of those suppressive drug to bacteria culture can reduce the cyclic-di-GMP level and lead to activation of the pspF-Clp and by-pass the RpfC/RpfG two-component system in theory .The result from rt-qPCR data below showed the above synthetic biological system was functional, indicated by the significant up-regulation of reporter mRNA.

### DSF treatment assay

![](https://static.igem.org/mediawiki/2019/3/33/T--Hong_Kong-CUHK--DSF_treatment.jpeg)

Figure 12: The graph showed the fold change in eforRed expression after various concentration of DSF treatment. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation. One-way ANOVA was used to analyse and determine the statistical significance of relative transcription level compared with control (induction time = 0). One star sign (\*) indicates significance at p < 0.05.

As DSF is the extracellular signaling molecule secreted by the *Xcm* bacteria, the aim of our product is to detect the existence of DSF and release color signal. The DSF treatment assay helped us ascertain whether the engineered *E. Coli* starts transcription under environment with DSF. Results indicated that at 1uM DSF concentration, eforRed transcript significantly increased in the bacteria, proving that the bacteria responsed to DSF molecules.

The cells were first induced for 4 hours by 1mM IPTG. Then, it was followed by the 16-hour treatment by 0-1000uM of DSF. The bar represent the mean of relative transcription level derived from replicates (n=3) and error bars represent the standard deviation.