iGEM 2017 — Team TMMU-China

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# Journal

**December**：We began to recruit team members to establish the 2017’s team, and team members consulted literature and widely read wikis in previous years to have a general understanding about iGEM.

**January**：We held quite a few times of brainstorms to discuss the potential project.

**March**：After a couple of months of learning and training, 14 team members were gathered to establish the TMMU-China team. Team registration was completed. And we set our project .

**April**：We attended iGEM Southwest Union and had an overall assessment of the safety and feasibility of our project.

**June**：The *B. subtilis* and *L. lactis* strain was constructed and the utility of it was tested.

**July**：We completed the optimizations of the system, and constructed the plasmid containing the transcription factor AimR and PlcR, signal peptide gene AimP and PapR. Some of the members set out to design and finish the mathematic modeling.

**August**：We attended the 2017 CCiC(Central China iGEM Consortium) held in Fujian Agriculture and Forestry University, receiving advice from Igem headquarter King L. CHOW and former iGEMer Zhang Haoqian. And together with SCU-WestChina , we cooperated in helding the exhibition in Chengdu Public Library, publicizing “GMO & Human Health”.

**September**：We completed the construction of all the 4 Parts.

**October-November**：We at last perfected our preparation for presentation, poster and wiki.

# Protocols

#### 1. Bacteria and culture media

Bacteria strains used in this study are NZ9000 (*L. lactis*) and DH5α (*E. coli*). DH5α was used as the cloning host. *E. coli* were cultured in Luria-Bertani (LB) medium (agar or broth) at 37 °C. *L. lactis* was cultured in M17GS (M17 broth supplemented with 0.5 % (wt/vol) glucose, 0.55 % (wt/vol) sucrose) medium at 30 °C (agar or broth). Kanamycin was used at the concentration of 50 μg/mL for *E. coli*. Erythromycin was used at the concentration of 20 μg/mL for *L. lactis*.

#### 2.Preparation for competent *B. subtilis* cells

Streak out the strain to be made competent on an LB or TBAB agar plate as a large patch and incubate overnight at 30 ℃. The following morning scrape off the cell growth off the plate and use to inoculate fresh, pre-warmed, SpC medium to give an OD600 reading of about 0.5. Incubate the culture at about 37℃ with vigorous aeration and take periodic OD readings to assess cell growth. When the rate of cell growth is seen to depart from exponential inoculate 200ml of pre-warmed, SpⅡmedium with 2ml of stationary-phase culture and continue incubation at 37℃ with slower aeration. After 90min incubation, pellet the cells by centrifugation at room temperature. Carefully decant the supernatant into a sterile container and save. Gently resuspend the cell pellet in 18ml of saved supernatant and add 2ml of sterile glycerol; mix gently. Aliquot the competent cells in sterile tubes, freeze rapidly in liquid nitrogen or a dry-ice bath and store at -70℃.

#### 3. Polymerase Chain Reaction

All PCR reactions were done with Fast Taq Mix DNA polymerase from Novoprotein Scientific Inc (Shanghai, China) and Primerstar Max DNA polymerase from Takara Bio Inc. (Dalian, China).

**(1). Fast Taq Mix DNA polymerase**

For colony PCR, Fast Taq polymerase is used. As the template, the colony of *E. coli* is picked from a plate and culture the *E. coli* when the medium become turbid. The component and cycling conditions are listed in the table below.

| 2×Fast Taq Mix(SinoBio) | | Cycling conditions | | |
| --- | --- | --- | --- | --- |
| Component Volume(μL) Tempreture(℃) Time Cycle | | | | |
| Premix Taq | 5 | 94 | 5 min | 1 |
| Bacteria solution | 1 | 94 | 30 sec | 35 |
| Primer F (20 μM) | 0.2 | 55 | 30 sec |
| Primer R (20 μM) | 0.2 | 72 | 1 kb/min |
| dd H2O | 3.6 | 72 | 5 min | 1 |
| Total | 10 | 16 | 1 min | 1 |

**(2). Primerstar Max DNA polymerase**

Primerstar Max DNA polymerase is used when high fidelity is needed. Primerstar Max has been used for amplifying DNA fragments and generating specific mutations in genes with the use of primers. The cycling program is listed in the table below.

| 2×PrimerStar Max(Takara) | | Cycling conditions | | |
| --- | --- | --- | --- | --- |
| Component Volume(μL) Tempreture(℃) Time Cycle | | | | |
| PrimerStar Max | 25 | 98 | 5 min | 1 |
| DNA Template | 1 | 98 | 10 sec | 35 |
| Primer F (20 μM) | 1 | 52 | 15 sec |
| Primer R (20 μM) | 1 | 72 | 10 sec/min |
| dd H2O | 22 | 72 | 5 min | 1 |
| Total | 50 | 16 | 1 min | 1 |

#### 4. Restriction enzyme digestion and T4 DNA ligation

**(1).Restriction enzymes used in this work were FastDigest enzymes (Thermo Scientific).**

Combine the following reaction components list at room temperature and the mixtures were incubated with recommended temperatures and time.

| Component Volume | |
| --- | --- |
| FastDigest Enzyme 1 | 2.5 μL |
| FastDigest Enzyme 2 | 2.5 μL |
| FastDigest Green Buffer | 5 μL |
| Plasmid or DNA fragment | up to 5 μg |
| dd H2O | add to 50μL |
| Total | 50 μL |

The digestion productions were purified by the Wizard SV Gel and PCR Clean-Up Kit (Promega Corporation).

**(2).DNA ligation used in this work was DNA Ligation Kit Ver.2.1 from Takara Bio Inc. (Dalian, China).**

Mix linearized plasmid vector DNA and a DNA fragment in a total volume of 2.5 μL. The amounts of vector: fragment is 0.03 pmol : 0.03 - 0.3 pmol. Add an equal volume of ligation mixture (2.5 μL) as the DNA solution and mix thoroughly. Incubate at 16 °C for 30 minutes.

#### 5. Seamless Cloning

For cloning our target fragments into vectors, NovoRec seamless cloning KIT from Novoprotein Scientific Inc. was used. Seamless cloning can simultaneously combine one or more PCR products with a linearized vector when the DNA to be joined shares 15 – 25 bp of homology at each end. The required homology can be easily generated by adding complementary sequence to the ends of the PCR primers. No additional treatment of the PCR fragment is required, such as restriction digestion, ligation, phosphorylation, or blunt-end polishing. The efficiency of NovoRec PCR Seamless Cloning is over 95 %.

**(1)**. The acquisition of the linearized vector by enzyme digestion (> 15 ng/μL).
**(2)**. A small sequence (15-25 bps) overlapped with the end of the cloning site will be added onto the insert through a PCR step.
**(3)**. Recombination reaction.

| Component Volume | |
| --- | --- |
| ddH2O | up to 20 μL |
| 5 × Buffer | 4 μL |
| Linearized cloning vector | 0.03 pmol |
| PCR products of insertions | 0.09 ~ 0.3 pmol |
| Enzyme | 2 μL |

The recommended amount of vector for recombination reaction is 0.03 pmol. The recommended amount of insertion for recombination reaction is 0.09 ~ 0.3 pmol.

**(4)**. Incubate the mixture at 37 °C for 20 min and transform it into *E. coli*.

#### 6.Preparation of chemically competent *E. coli* cells

Inoculate 2ml LB broth with an aliquot (about 50ul)of the desired *E. coli* from the -80℃ freezer stock of cells. Incubate for 2h at 37℃. Add the 2ml seed culture to 250ml LB broth and grow at 37℃, shaking (about 200rpm) until OD600 of 0.3-0.4 (about 5 hours).Pre-cool the 50ml polypropylene tube, 80 EP tubes, CaCl2-glycerine (0.1mol/L CaCl2) and CaCl2- MgCl2 (80mmol/L MgCl2, 20mmol/L CaCl2). Set the centrifuge and prepare the ice tray. Transfer the bacteria into the 50ml polypropylene tube. Place it on ice for 10 minutes. Centrifuge at 4℃, 4100rpm for 10 minutes. Discard supernatant, then place the tube upside down to make sure trace liquid medium runs out. Add 30ml of pre-cooled CaCl2- MgCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet. Centrifuge at 4℃, 4100rpm for 10 minutes. Discard supernatant then place the tube upside down to make sure trace liquid medium runs out. Add 2ml of pre-cooled CaCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet. Transfer to EP tubes (50ul every tube) and store at -80℃.

#### 7. Transformation of *E. coli*

Example Protocol: Standard heat-shock transformation of chemically competent bacteria.

Take competent cells out of -80°C and thaw on ice (approximately 20-30min). Take agar plates (containing the appropriate antibiotic) out of 4°C to warm up to room temperature or place in 37°C incubator. Mix 1 to 5μl of DNA (usually 10pg to 100ng) into 20-50μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times. Note: Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. Place the competent cell/DNA mixture on ice for 20-30min. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 seconds (45sec is usually ideal, but this varies depending on the competent cells you are using). Put the tubes back on ice for 2 min. Add 250-500μl LB or SOC media (without antibiotic) and grow in 37°C shaking incubator for 45min. Note: This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded on the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances. Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic. Note: We recommend that you plate 50μL on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants. Incubate plates at 37°C overnight.

#### 8.Transformation of *B. subtilis*

Thaw competent cells rapidly by immersing frozen tubes in a 37 ℃ water bath. Immediately, add one volume of SpⅡEGTA to the thawed cells; mix gently. In a sterile test tube add competent cells to the DNA solution and incubate in a roller drum at 37℃. Dilute the transformed cells as appropriate in T Base containing 0.5% glucose and plate immediately onto selective media.

#### 9. Transformation of *Lactococcus lactis*

**(1) Preparation of competent *L. lactis* cells**

To obtain competent cells, the cultures were grown to an optical density at 600 nm of 0.5 to 0.8 and then diluted 100-fold in SGM17 (M17GS containing 0.5 M sucrose) supplemented with glycine. After growth at 30 °C to an optical density at 600 nm of 0.2 to 0.4, the cells were harvested by centrifugation at 4 °C at 5,000x g. Following two washes in ice-cold 0.5 M sucrose containing 10% glycerol, the cells were suspended in 1/100 culture volume of washing solution and then stored in aliquots at -85 °C until use.

**(2) Transformation by electroporation**

The cell suspensions were thawed on ice. Portions (40 μL) were mixed with 1 μL of DNA and then transferred to an ice-cooled electroporation cuvette (2-mm electrode gap) and exposed to a single electrical pulse. The pulse was delivered by a Gene-Pulser (Bio-Rad, Calif.) set at 25 μF and normally at 2.0 kV. The cuvette was connected in parallel to a 200-Ω resistor (pulse controller; Bio-Rad), resulting in time constants of 4.5 to 5ms.

**(3) Incubation and plate**

Immediately following the discharge, the suspensions were mixed with 0.96 ml of ice-cold SGM17MC (SGM17 containing 20 mM MgCl2 and 2 mM CaCl2) and left on ice for about 5 min. Appropriate dilutions were then made in SGM17MC, and the cells were incubated at 30°C for 2h. The plates M17GS contained 20 μg/mL of erythromycin for the selection of erythromycin-resistant transformants. Transformants were enumerated after 2 days of incubation at 30°C.

#### 10.Amylase activity analysis

Apply iodine on the plate with 0.1% starch and spectinomycin. Incubate the engineered bacteria and non-engineered bacteria onto the plate at 37℃. See if there are hydrolysis circles on the plate.

#### 11.Agarose Gel Electrophoresis

Weigh agarose powder and TAE buffer according to a proper portion, and add them to a 100ml conical flask (we usually make 1.5% Agarose Gel). Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave). Let the solution cool down to about 40-50℃ and add DNA gel stain (usually we use EB), pour the solution into the gel casting tray with appropriate comb. Let the gel cool until it becomes solid. Pull out the comb carefully. Place the gel in the electrophoresis chamber. Add enough TAE Buffer so that there is about 2-3mm of buffer over the gel. Pipette DNA samples mixed with appropriate amount of DNA loading buffer (the dye/GeneFinder is in the loading buffer) into wells on the gel. Run the gel at 135V for about twenty minutes.

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Contact us
Email: igem@tmmu.edu.cn
Address:Third Military Medical University, No.30 Gaotanyan Street Shapingba District, Chongqing, P.R.China 400038

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# TMMU-China

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:TMMU-China/Results

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# Results

### Construction of the recombinant plasmids

The Aim sequence was synthesized. After that, we cloned the AimR-AimP-pAimX fragment using PCR. We also cloned the GFP sequence by PCR. The two fragments were purified and assembled with the enzyme digested pDG1730 plasmid. The pDG1730 plasmid is used to knock the composite part into the genome of *B.subtilis*. The *amyE* gene upstream and downstream sequences were used to target the composite part into the amyE locus. The Spc gene confers resistance to spectinomycin.

![](https://static.igem.org/mediawiki/2017/c/c6/T--TMMU-China--7_%E6%9E%AF%E8%8D%89%E8%8A%BD%E5%AD%A2%E6%9D%86%E8%8F%8C.jpg)

Fig The construct map of pDG1730

### Construction of the composite part knock-in *B.subtilis* strains

We used natural transformation to transform the B.subtilis 168 strain. The competent cells of B.subtilis 168 strain were induced using nutrient-limited medium. The knock-in strains were selected by 100 mg/mL spectinomycin. The genomic DNA of the knock-in strains were extracted and used as the PCR template. Specific primers were used to conduct PCR to confirm the integration of the composite part. Here are some examples of the confirmation results.

### Expression of the GFP reporter in the knock-in strains

We also incorporate the pDG1730 vector into the B.subtilis 168 strain. Compared to this reference strain, we found that the pAim-AimR-AimP-pAimX-GFP composite part can emit green fluorescence. However, to our surprise, the pVeg-AimR-AimP-pAimX-GFP composite part can not emit green fluorescence. The phage genes are usually highly packed. In the promoter region of the AimR genes, some regulatory elements might be important for the expression of composite part, thus the pVeg promoter can not control the expression of AimR genes. The exact mechanisms are not known.

![](https://static.igem.org/mediawiki/2017/1/10/T--TMMU-China--8_%E6%9E%AF%E8%8D%89%E8%8A%BD%E5%AD%A2%E6%9D%86%E8%8F%8C.jpg)

Fig Characterization of the pAim-AimR-AimP-pAimX-GFP composite part

### Measurement of fluorescence curve along time

To explore the dynamics of the auotoinhibition of the synthetic AimR-AimP system, we track the green fluorescence intensity of the pAim-AimR-AimP-pAimX-GFP knock-in strain along time using the Microplate Reader. As shown in the figure, we found that the expression of GFP increased steadily to a peak, and then the green fluorescence intensity decreased and finally reached a plateau. This result is consistent with the autoinhibition prediction of the pAim-AimR-AimP-pAimX-GFP composite part.

![](https://static.igem.org/mediawiki/2017/b/b9/T--TMMU-China--9_%E6%9E%AF%E8%8D%89%E8%8A%BD%E5%AD%A2%E6%9D%86%E8%8F%8C.jpg)

Fig. Fluorescence curve along time.

### Selection of the appropriate Agr system from the four Agr types

There are four different classes in the Agr systems which are referred to as Agr-I, Agr-II, Agr-III, and Agr-IV. Among the four classes of AIPs, the five-residue thiolactone ring structure is always conserved, while the other ring and tail residues differ. Similarly, the proteins involved in signal biosynthesis (AgrB and AgrD) and surface receptor binding (AgrC) also show variability. The activity of the four Agr classes is varied. To select the appropriate Agr system for engineering, we first conduct the hymolysis experiment with four *S. aureus* strains containing different Agr classes.

Table 1 The strains used and their cognate Agr groups

|  |  |
| --- | --- |
| Agr groups | Strain Name |
| Agr-I | Newman |
| Agr-II | N315 |
| Agr-III | MW2 |
| Agr-IV | XQ |

Production of exoproteins such as hemolysins in *S. aureus* is controlled by the agr system. Different Agr types are usually associated with different hemolytic activity. To establish the link between them, we placed four *S. aureus* strains from Agr Ⅰ to Agr Ⅳ on rabbit blood agar plate (Fig. 2). The hemolytic activity can be clearly observed from the rabbit blood agar plate. The strain of Agr Ⅰ and Agr Ⅳ types show visible and very transparent hemolysis zone around the bacteria, indicating the hemolytic activity of strain of Agr Ⅰ and Agr Ⅳ are very strong. On the other hand, no hemolysis zone of Agr Ⅱ is observed. The hemolysis zone of Agr Ⅲ is very small. The larger the hemolysis zone, the higher activity of the Agr system. AIP-I and AIP-IV differ by only one amino acid, they are grouped together and can function interchangeably. From this result, we are going to use the Agr Ⅰsystem to do the following experiments.

Interestingly, different AIP signals cross-inhibit the activity of the others. For example, group I *S. aureus* quorum sensing can be activated by AIP-I but is inhibited by the AIPs produced by group II or III *S. aureus* trains. From the engineering perspective, the AIP-II or AIP- III sender cell can be used to inhibit the Agr I Receiver cell once it is activated. Since AIP-II displays large variation from AIP-I, we are going to develop an AIP-II Sender cell. The AIP-II Sender cell can also be used to inhibit the virulence of Agr group I *S. aureus*.

![](https://static.igem.org/mediawiki/2017/8/82/T--TMMU-China--3%E4%B9%B3%E9%85%B8%E4%B9%B3%E7%90%83%E8%8F%8C.png)

Fig. 2 Four *S. aureus* strains from Agr I to Agr IV on rabbit blood agar plate.

### Characterization of the P2-GFP composite part in *S. aureus*

In addition to the hemolysis zone phenotype, we want to establish another phenotype to reveal the activity of the Agr system. In the Agr locus, two promoters P2 and P3 were found to the direct target of the active AgrA transcription factor. Place the GFP gene under the promoter of P2 or P3 can be used as the reporter of the Agr system. The 2007 Cambridge Team constructed the P2-GFP composite part. They intended to test the part in E. coli; however they failed to do so. To characterize whether this P2-GFP part can function also in the Gram-positive strain, we test this composite part directly in *S. aureus*. The P2-GFP composite were cut from the pSB1C3-P2-GFP part, then it was ligated into the shuttle vector pLI50. After that the pLI50-P2-GFP was transformed into the *S. aureus* strain RN4220. We found that the RN4220::pLI50-P2-GFP strain show strong green fluorescence compared to the RN4220::pLI50 strain on plate (Fig. 3). This data suggest that the P2-GFP composite can be functional when the Agr system is present. We want further to know whether this part can be used to reveal the varied activity of the four Agr types, we transformed the pLI50-P2-GFP construct from RN4220 to Newman(Agr typeⅠ), N315 (Agr type Ⅱ). We found that the Newman::pLI50-P2-GFP strain emitted strong green fluorescence, however, barely detectable green fluorescence can be observed of the N315::pLI50-P2-GFP strain. The expression of P2-GFP in Newman and N315 is consistent with their hemolysis activity on the blood agar plate. These data collectively confirmed that the P2-GFP composite part can be used to track the activity of the Agr system.

![](https://static.igem.org/mediawiki/2017/5/59/T--TMMU-China--5_%E4%B9%B3%E9%85%B8%E4%B9%B3%E7%90%83%E8%8F%8C.jpg)

Fig. 3 White light (A) or fluorescence (B) of RN4220::pLI50-P2-GFP strain on plate.

### Measurement of fluorescence curve along time

To explore the dynamics of the autoinduction of the synthetic AIP system, we track the green fluorescence intensity of the RN4220::pLI50-P2-GFP strain along time using the Microplate Reader. As shown in the figure, we found that the expression of GFP increased steadily, and finally reached a plateau (Fig. 4). This result is consistent with the autoinduction prediction of the composite part. The same fluorescence changes along time have been observed in Newman::pLI50-P2-GFP (data no shown).

![](https://static.igem.org/mediawiki/2017/a/aa/T--TMMU-China--4%E4%B9%B3%E9%85%B8%E4%B9%B3%E7%90%83%E8%8F%8C.jpg)

Fig. 4 Fluorescence curve along time

### Construction of the recombinant plasmids used in L. lactis

Erythromycin resistance has been reported to be a useful selectable marker in E. coli or L. lactis. However, in our hands, erythromycin selection was usually problematic in E. coli, giving rise to high background levels of untransformed cells. Since selection for kanamycin resistance is much more efficient in E. coli, kanamycin resistance gene with promoter from pEASY was cloned in pMG36e EcoRI site by seamless clone. The resulted plasmid, pMG36ek, contains ampicillin- and erythromycin-resistance (Fig. 5).

![](https://static.igem.org/mediawiki/2017/b/bc/T--TMMU-China--7_%E4%B9%B3%E9%85%B8%E4%B9%B3%E7%90%83%E8%8F%8C.jpg)

Fig. 5 pMG36ek Map.

The Agr system was divided into two parts, AIP-producing sender and AIP receiver, which constructed sender/receiver synthetic communication system.

The agrB-agrD fragment was cloned at the downstream of P32 promotor in pMG36ek by seamless clone. The resulted plasmids were named pMG36ek-BD-I (from Agr-I), pMG36ek-BD-II (from Agr-II), and pMG36ek-BD-IV (from Agr-IV). Unfortunately, we didn’t get the pMG36ek-BD-III (from Agr-III) plasmid, maybe BD from Agr-III is toxic to cell. Then, the pMG36ek-BD was transformed into the L. lactis strain NZ9000. The constructed NZ9000::pMG36ek-BD-I, NZ9000::pMG36ek-BD-II, and NZ9000::pMG36ek-BD-IV were the AIP-producing sender.

To test whether the AIP receiver work or not, gfp gene with P2 promoter was cloned at the EcoRI site of pMG36ek, which produced pMG36ek-P2-GFP (Fig. 6). If the agrC-agrA fragment was cloned at the downstream of P32 promotor in pMG36ek, the expressed protein AgrC and AgrA will receive the signal of corresponding AIP, and induce gfp gene at the downstream of P2 promoter. Unfortunately, maybe the agrC-agrA fragment was toxic to E. coli and L. lactis, and we cannot get the plasmid. We also used various of promoter, such as P2 or PnisZ (nisin inducible promoter in NZ9000), to change the promoter of agrC-agrA fragment, and we still not get the corresponding vectors. Then, the *S. aureus* (strains from Agr I to Agr IV) or *S. aureus* carrying pLI50-P2-GFP plasmid were used as AIP receiver.

![](https://static.igem.org/mediawiki/2017/2/2f/T--TMMU-China--6_%E4%B9%B3%E9%85%B8%E4%B9%B3%E7%90%83%E8%8F%8C.jpg)

Fig. 6 pMG36ek-P2-GFP Map.

### Test the AIP-producing Sender/receiver synthetic communication system cell with *S. aureus*

To test the sender/receiver synthetic communication system work or not, two tests have been done. Different AIP signals cross-inhibit the activity of the others. For example, AIP-I will activate group I Agr system and inhibited group II or III Agr system, and the most significant phenomenon change of inhibition is the change of hemolysis zone on rabbit blood agar plate. Therefore, *S. aureus* strains Agr I was co-cultured with NZ9000 or NZ9000::pMG36ek-BD-I/II/IV on rabbit blood agar plate, but the hemolysis zone of each co-culture colony has not significant changes (Fig. 7). The same phenomenon changes have been observed in *S. aureus* (strains from Agr II to Agr IV) colonies co-cultured with NZ9000::pMG36ek-BD-II and NZ9000::pMG36ek-BD-IV (data no shown). To further assess our sender/receiver synthetic communication system, stationary-phase cultures of *S. aureus* (strains from Agr I to Agr IV) carrying pLI50-P2-GFP plasmid and NZ9000::pMG36ek-BD-I/II/IV were spotted onto M17GS agar separately, their centers were separated by ~8 mm. If the AIP signal was produced, when sender and receiver cells were spotted directly adjacent to one another, we will observe fluorescence changes from the receiver cells at the edge of the colony closest to the sender cells. But we didn’t observe any significant changes of the receiver cells (*S. aureus* strains from Agr I to Agr IV, data not shown). These two tests indicated that, the sender (NZ9000::pMG36ek-BD-I/II/IV) did not produce functional AIP signals.

![](https://static.igem.org/mediawiki/2017/8/83/T--TMMU-China--8_%E4%B9%B3%E9%85%B8%E4%B9%B3%E7%90%83%E8%8F%8C.jpg)

Fig. 7 Four S.aureus (strains from Agr I to Agr IV) co-cultured with NZ9000 (A) or NZ9000::pMG36ek-BD-I (B) co-culture on rabbit blood agar plate.

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