BBa_K1949060 meets the Silver Medal criteria!

We simulated our final genetic circuits and found that the circuits would not work, because Prhl (BBa_I14017) strength was too weak. (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 team Tokyo_Tech, but we noticed that they were inappropriate for two reasons (see our work Rhl system assay). Then, we decided to further improve this Prhl and obtain our original improved Prhl (included in BBa_K1949060) that suited our goal. Our purpose is to create a strong Prhl for our final genetic circuits. This experiment consists of the three parts below.

For more information, read this page !

1. Improvement of iGEM 2014 team Tokyo_Tech's Prhl(BBa_K1529300, BBa_K1529310) and characterize RhlR assay (Fig.4-3-1)




Fig. 4-3-1 Effect of RhlR by tagging LVA
and The colonies of transformants with a rhlR (left) or a rhlR - LVA (right)

Improvement of the wild type Prhl(BBa_I14017) (Fig.4-3-2)




Fig. 4-3-2 RFU of GFP / Turbidity of imoroved Prhl mutants

Comparison of iGEM 2014 team Tokyo_Tech's Prhl to our original improved Prhl (included in BBa_K1949060) (Fig.4-3-3)




Fig. 4-3-3 Comparison of Prhl(NM) by Prhl(LR)

The past improved Prhl did not suit for our final circuits and we could construct the improved Prhl appropriate to our final circuits.

Name	Type	Description	Design	Length(bp)
BBa_K1949001	Measurement	Pcold-gfp	Yoshio Takata	1033
BBa_K1949032	Composite	PBAD-rbs-yafO	Yoshio Takata	1638
BBa_K1949033	Composite	PBAD-rbs-yafO-tt-Ptet-rbs-gfp	Yoshio Takata	2580
BBa_K1949052	Composite	PBAD-rbs-amiE	Yoshio Takata	2712
BBa_K1949060	Composite	Prhl(NM)-rbs-gfp	Yoshio Takata	799
BBa_K1949100	Composite	Plac-rbs-mazE	Yoshio Takata	565
BBa_K1949101	Composite	PBAD-rbs-mazF	Yoshio Takata	1575
BBa_K1949102	Composite	PBAD-rbs-mazF-tt-Ptet-rbs-gfp	Yoshio Takata	2520
BBa_K1949103	Composite	Ptet-rbs(BBa_B0034))-mazE	Yoshio Takata	332
BBa_K1949104	Composite	Ptet-rbs(BBa_J61117)-mazE	Yoshio Takata	332

BBa_K1949032 meets the Silver Medal criteria!

YafN is an antitoxin corresponding to toxin YafO, and YafN reverses inhibition of cell growth. yafN and yafO exist as an operon, and YafN also functions as a repressor in the operon. Therefore, yafNO operon’s translation is autoregulated by antitoxin YafN. When Lon protease senses environmental stresses such as DNA damage, the operon becomes active because of degradation of YafN by Lon protease.

Confirming Yafo Function as Toxin on Agar Medium

We first confirmed YafO function by observing formation of colonies on agar plate. This experiment was carried out using E. coli which inducible yafO expression by arabinose and E. coli without yafO gene. Construction of plasmids used in this experiment is shown in below. We inoculated four types of E. coli differently on agar plate with and without arabinose. As a result(Fig. 4-3-4), all types of E. coli were able to form colonies on agar plate with no arabinose, and colonies of E. coli (c) and (d) had RFU (Relative Fluorescence Units). On the other hand, E. coli (a) and (c) couldn’t form any colonies on agar plate containing arabinose. From these results, we confirmed that all types of E. coli in below functions as we expected.

Toxin-antitoxin assay

From previous experiment, we were able to confirm that YafO works as toxin. Next, we confirmed whether the cell growth reverses by antitoxin YafN after inhibition by YafO. Construction of plasmids used in this experiment is shown in below. We prepared E. coli which can induce expression of yafO by arabinose and YafN by lactose. As comparisons, we also carried out same experiment with E. coli containing no yafO gene, no yafN gene and none of them. As a result(Fig. 4-3-5), we couldn’t observe cell growth reversing after adding IPTG to induce expression of yafN, and their turbidity became constant as E. coli (c) containing no yafN. In this experiment, YafN couldn’t reverse the cell growth after inhibition by YafO. It seems to be difficult to control cell growth just like mazEF system, which demonstrated reverse of cell growth by antitoxin after function of toxin.

For more information, read BBa_K1949020, BBa_K1949030 !

BBa_K1949100, BBa_K1949101 and BBa_K1949102 meets the Bronze Medal criteria!

BBa_K1949102 is our Best Composite Parts !

Stop & Go

The biggest attraction of the TA system is that it is able to control cell growth and synthesis of protein. In this experiment, mazE expression was induced by the addition of IPTG(2 mM) after mazE expression was induced by the addition of arabinose(0.02%). As a result, it was able to resuscitate from a state of being inhibited cell growth. We named this experiment as "Stop & Go" because it was to resuscitate growth from inhibiting cell growth.

It was found from Fig. 4-3-5 that MazF inhibited cell growth. MazE was induced 2 h after maze expression, and about 8 h later, cell growth was recovered that had stopped. From these results, it was suggested that E. coli whose cell growth was inhibited by MazF was able to resuscitate by expression of mazE.

Go & Stop

We found that a toxin inhibits cell growth, and an antitoxin resuscitates it. However, what will happen when a toxin is expressed after the antitoxin constitutive expression? Therefore, we conducted the experiment. Since cell growth was resuscitated after cells had grown, we named this experiment, "Go & Stop".

From Fig.4-3-6, E. coli encoded mazE which is on downstream of weak RBS(BBa_J61117) has more turbidity than E. coli encoded mazE which is on downstream of normal RBS(BBa_B0034). Both of those E. coli would reach stationary phase when there is little RFU of GFP(Fig. 4-3-6). E. coli encoded mazE which is on downstream of normal RBS reached almost the same stationary phase as E. coli without TA system.

For more information, BBa_K1949101, BBa_K1949102, BBa_K1949103 and BBa_K1949104.