Description

      This year, we have improved 2 previous projects. The first one is the project of 2016 BIT-China, and another one is the project of 2017 CMUQ. We have improved 3 BioBricks, BBa_K2570020, BBa_K2570021, BBa_K2570022 and sent the improved parts to iGEM headquarters in October, 2018.

Improvement of 2016 BIT-China and 2017 CMUQ

      To inhibit the engineering bacteria in short time, the 2016 BIT-China iGEM Team chose to construct a controllable circuits with toxin genes mazF . And here they redesigned circuits of araC+pBAD+B0032+mazF BBa_K2120002 to verify its function. We are curious about the effects of different promoters on the expression of toxin proteins and we further explore and created a new BioBrick Part BBa_K2570021, which has a functional improvement upon BBa_K2120002.
At the same time, our team have added new, high quality experimental characterization data to the wildtype promoter sequence of the E.coli proU operon from 17 CMUQ which functions as an osmolarity sensor (BBa_K2467001), obtained and uploaded the detailed data according to the requirements.

Construction of the mazF expression system combined with proU promoter

      Firstly, we explored the effect of toxin protein expression under the control of the salt-controlled promoter. We set different salt concentrations as experimental variables, and the green fluorescent protein was used as the reporter.
      Part: BBa_K2570020 proU+B0034+GFP+B0032+mazF

Fig 1. (A) A schematic diagram of a salt-controlled promoter expressing the toxin protein (mazF) circuit.
(B) Fluorescence levels per OD at different salt concentrations.
(C) Fluorescence images at different salt concentrations.

      We set the salt concentration 0mm, 50mm, 100mm, 300mm, 500mm (NaCl). In Fig. 1, at the same OD value, the measured GFP fluorescence value increased with increasing salt concentration. In addition, green fluorescence at different salt concentrations can be visually observed, and it is concluded that an increase in salt concentration before 300mM NaCl enhances the expression of a salt-controlled promoter, an increase in the expression of green fluorescent protein, and an increase in GFP fluorescence value. At a salt concentration of 300 mM NaCl to 500 mM NaCl, the fluorescence intensity at the unit OD value is weakened. We speculate that higher salt concentrations have an adverse effect on cell growth and salt-controlled promoter expression.
In the experiment, the ideas of quantification and correction of Interlab guided our data analysis.

Construction of the mazF expression system combined with dsrA promoter

      Secondly,we explored the effect of toxin protein expression under the control of a temperature promoter. We set different temperature as experimental variables, and we added green fluorescent protein for characterization.
      Part: BBa_K2570021 dsrA+B0034+GFP+B0032+mazF

Fig 2. (A) Schematic diagram of a temperature controlled promoter expressing the toxin protein (mazF) circuit.
(B) Fluorescence levels per OD at different salt concentrations.
(C) Fluorescence images at different salt concentrations.

      We set the temperature concentration 25℃, 30℃, 35℃, 40℃. In Fig. 2, at the same OD value, the measured GFP fluorescence value increased with increasing culture temperature. In addition, the green fluorescence at different culture temperature can be visually observed, and it is concluded that an increase in culture temperature enhances the expression of a temperature controlled promoter, an increase in the expression of green fluorescent protein, and an increase in GFP fluorescence value. At the culture temperature of 40 degrees, the fluorescence intensity of the unit OD value was significantly weakened. We speculated that the higher culture temperature has an adverse effect on cell growth and temperature-controlled promoter expression.

Precise comparison

      The araC+pBAD promoter can be controlled tightly by arabinose. We transformed the plasmid consisting of araC+pBAD+B0032+mazF (BBa_K2120002) in E.coli. MG1655 and induced expressions with different concentrations of arabinose.
      Furthermore, the lethal efficiency of the toxin protein can only be roughly obtained by measuring the OD values. We have obtained more accurate experimental data through bacteriostatic experiments under different conditions.

Fig. 3 Point board data map. Reflects the lethal state of the toxin protein.

      Using bacteriostatic experiments, we obtained more accurate experimental data compared with the lethal efficiency of BBa_K2120002. Under the condition of adding arabinose concentration of 50% and reaction time of 150 min, we can achieve the best lethal efficiency, and it is expected to be applied as a suicide switch in futher environmental projects. In addition, we obtained the sequence of wild-type mazf toxin protein from E. coli and utilized it as a positive control.
      Finally, in this experiment, interlab's quantification and correction ideas guided our data analysis.