When we tried to represent Snow White with TA system and Quorum Sensing, we faced two problems. First, we could access too little data of TA system to work on our project. Second, simulation of our final genetic circuits revealed that Rhl system was too weak to function as well as Lux system and our final circuits could not work well. So we decided to characterize the existing TA system and rhlR parts, and improve the past improved Prhl in order to represent Snow White as faithfully as we could. As a results, the data we obtained makes easy to use these existing TA system and rhlR parts. Furthermore, our original improved Prhl expands the possibility to use Quorum Sensing.

1-1. "Background”for our Characterization of TA system

TA system is the function that regulates cell growth on the genomic DNA of many microorganisms. So far, iGEM 2014 team ULB-Brussels, iGEM 2013 team UGent had studied about ccdB-ccd, iGEM 2014 team Wageningen UR about Kid-Kis and Zeta-Epsilon, iGEM 2015 team UMaryland about Hok-Soc. Almost all the iGEM teams dealing with TA system aimed to use it as Kill Switch or substitution for antibiotics. On the other hand, we aim to study the interaction of toxin and antitoxin in a cell. Besides some teams failed to show enough data of TA system assay. Although UCSF 2012 experienced the communication through TA system including mazEF system, they did not introduce toxin and antitoxin into a cell and could not show mazEF system assay data. Our results show that the cell growth can be controlled by mazEF system.

iGEM 2016 team Tokyo_Tech has studied about mazEF system (MazF : toxin , MazE : antitoxin) that has been researched well as TA system.

1-2. Summary of the experiment

We studied mazEF system with various conditions and found that the best condition of expressing mazEF system in one cell. Besides, we also found that cell growth regulated by the alternative expression of MazE and MazF.

1-3. Results

1-3-1. Adjustment of Expression of MazF

It was concluded that MazF should be induced under the condition that the arabinose concentration is 0.02% so that MazF functions as a toxin and GFP fluoresces sufficiently in mazEF system assay.

E. coli A : Pcon- rbs - gfp (pSB6A1) , Plac - rbs (pSB3K3)
E. coli B : PBAD- rbs - mazF (pSB6A1) , Plac - rbs (pSB3K3)

Fig.1-3-1-1 Relative value of Turbidity, Relative Fluorescence Units (RFU) of GFP and RFU of GFP / Turbidity of medium where E. coli was cultured vs concentration of arabinose. E. coli A and E. coli B were cultured in the presence of indicated amounts of arabinose, and turbidity (upper graph), RFU of GFP (middle graph), and RFU / turbidity (lower graph) were measured. Also, the same cells were cultured in the absence of arabinose, and measurements were performed similarly as above. The ratio of the former values to the latter values were calculated.

1-3-2. mazEF System Assay

Since MazE resuscitates E. coli after MazF expression inhibits cell growth and translation, MazF inhibits cell growth and translation after MazE expression, it was suggested that using mazEF system can regulate cell growth and translation.

E. coli B : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Plac-rbs (pSB3K3)
E. coli C : PBAD - rbs (pSB6A1) , Plac-rbs (pSB3K3)
E. coli D : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Plac-rbs - mazE (pSB3K3)

Fig.1-3-2-1 Time vs Turbidity (Stop & Go)

E. coli A : Pcon - rbs - gfp (pSB6A1) , Plac-rbs (pSB3K3)
E. coli B : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Plac-rbs (pSB3K3)
E. coli D : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Plac - rbs - mazE (pSB3K3)

Fig.1-3-2-2 Time vs RFU of GFP (Stop & Go)

E. coli C : PBAD - rbs (pSB6A1) , Plac-rbs (pSB3K3)

E. coli E : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , vector (pSB3K3)
E. coli F : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Pcon - rbs - mazE (pSB3K3)
E. coli G : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Pcon - weak rbs - mazE (pSB3K3)

Fig.1-3-2-3 Time vs Turbidity (Go &Stop)

E. coli A : Pcon - rbs - gfp (pSB6A1) , Plac-rbs (pSB3K3)
E. coli E : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , vector (pSB3K3)
E. coli F : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Pcon - rbs - mazE (pSB3K3)
E. coli G : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Pcon - weak rbs - mazE (pSB3K3)

Fig.1-3-2-4 Time vs RFU of GFP (Go &Stop)

E. coli A : Pcon - rbs - gfp (pSB6A1) , Plac-rbs (pSB3K3)
E. coli F : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Pcon - rbs - mazE (pSB3K3)
E. coli G : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Pcon - weak rbs - mazE (pSB3K3)
E. coli B : PBAD - rbs - mazF - tt - Pcon - rbs - gfp (pSB6A1) , Plac-rbs (pSB3K3)

Fig.1-3-2-5 Translation efficiency of each E. coli.

1-4 Discussion

We learned the appropriate arabinose concentration, 0.02%, to induce MazF. This assay firstly shows that in one cell, MazE resuscitates E. coli after MazF expression inhibits cell growth and translation, then, MazF inhibits cell growth and translation after MazE expression. It is suggested that using mazEF system regulate cell growth and translation. In conclusion, we can indicate, as the function of TA system, not Kill Switch but a regulator of systems. It will be useful for information processing.

2-1. "Background”for our improvement of Prhl

We simulated our final genetic circuits and found that the circuits did not work, because Prhl activity was too weak compared to Plux. (see the Model page and the AHL Only Assay page). We therefore considered using the improved Prhl (BBa_K1529300, BBa_K1529310) established by iGEM 2014 Tokyo_Tech, but we noticed that they were inappropriate for two reasons: crosstalk to C12 and type of rhlR (written below). Then, we decided to improve wild type Prhl(BBa_R0071).

2-2. Summary of the experiment

By introducing a single point mutation into wild type Prhl (BBa_R0071) by PCR, we obtained 198 Prhl mutants and obtained the Prhl mutants of which promoter activity was stronger than wild type Prhl(BBa_R0071), and named it Prhl(Noticeable Mutant) (BBa_K1949060), hereafter referred to as Prhl(NM), which suited our goal.

2-3. Results

The SN ratio of Prhl(NM)(BBa_K1949060) was higher than that of Prhl(LR)(BBa_1529310)and Prhl(NM)(BBa_K1949060) did not crosstalk to C12 unlike Prhl(LR)(BBa_1529310).

2-4. Discussion

Many iGEM teams used AHL and probably this tendency continues. When you use AHL, you will find that promoter strength is quite different depending on the type of AHL and it bothers you to design genetic circuits, for example, including Plux and Prhl. Prhl(NM) (BBa_K1949060) has almost as same strong activity as Plux. Furthermore, Prhl(NM) (BBa_K1949060) has higher specificity to C4 than Prhl(LR)(BBa_1529310). Prhl(NM) (BBa_K1949060) can be said that it expands the possibility of using various combination promoters depending on AHL.

3-1. "Background”for our Characterization of rhlR

When we used two type of rhlR, one was LVA-tagged and the other was normal, we found that gap introduced with rhlR barely expressed but gfp introduced with rhlR-LVA expressed greatly. We decided to find this cause.

3-2. Summary of the experiment

Measure RFU of GFP of the E. coli containing (a) Pcon-rbs-rhlR-LVA (pSB6A1), Prhl(LR)-rbs-gfp (pSB3K3), (b) Pcon-rbs-rhlR (pSB6A1), Prhl(LR)-rbs-gfp (pSB3K3), (c) Pcon-rbs-rhlR-LVA (pSB6A1), Prhl(RL)-rbs-gfp (pSB3K3) or (d)Pcon-rbs-rhlR(pSB6A1), Prhl(RL)-rbs-gfp (pSB3K3). And we found that gfp expresses much if it introduced with rhlR-LVA even though using stronger Prhl than wild type Prhl(BBa_R0071). Thls is because that RhlR, which represses Prhl without C4, tagged LVA is prone to be degraded and the concentration of RhlR in a cell decreases then the repression becomes weak.

3-3. Results

The level of GFP expression of (a)~(d) are shown below.

3-4. Discussion

The expression level of the gene under Prhl depends on not only Prhl activity but also the type of the rhlR. In other words, this characterize shows that the combinations of Prhl and rhlR can control the expression level of the gene under Prhl. It gives you more choices which Prhl and rhlR you use.