Difference between revisions of "Part:BBa K2570021"

Revision as of 16:24, 17 October 2018

dsrA+B0034+GFP+B0032+mazF

This is an improved version of BBa_K2120002

Temperature is one environmental variable that all organisms must deal with. Besides the direct effect of temperature on enzymatic reactions, temperature response involves remodeling of the expression pattern of genes, affecting transcription, RNA stability, translation efficiency, and/or proteolysis. RpoS is a stationary-phase and stress response sigma factor in E. coli and many other bacteria. One environmental cue that increases RpoS synthesis is low temperature (below 37°C). This increase is completely dependent upon dsrA, a small noncoding RNA[2]. dsrA was shown to stimulate RpoS translation by pairing with a portion of the mRNA upstream of the RpoS translation start that can pair with and occlude the ribosome-binding site of the transcript [3]. dsrA is more abundant in E. coli at low growth temperatures than at higher temperatures, resulting in the increased RpoS expression at low temperatures [4]. Temperature affects both the synthesis and the stability of dsrA, leading to thermocontrol of RpoS translation.

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.

Then,we use the GFP BBa_E0040 to characterize the intensity of the temperature-controlled promoter.

Figure.(A) schematic diagram of a temperature controlled promoter expressing the toxin protein (mazF) circuit.

Figure.(B) Fluorescence levels per OD at different salt concentrations.

Figure.(C) Fluorescence images at different culture temperature.

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

In addition, we transformed the araC+pBAD+B0032+mazF (BBa_K2120002) plasmid and set different concentrations of arabinose for induction expression. The araC+pBAD promoter can be controlled tightly by using arabinose.

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

On the other hand, by measuring the OD value, the lethal efficiency of the toxin protein can only be roughly obtained. We obtain experimental data through more accurate experimental means for the control experiment design.

Using experimental methods different from the measured od value, we obtained more accurate experimental data, compared with the lethal efficiency of BBa_K2120002. Under the best conditions, our improved parts can achieve good lethal efficiency, and it is expected to be applied as a suicide switch in environmental projects.

Figure.Protein modeling.

PS: We obtained the wild-type mazf toxin protein sequence from E. coli and conducted experiments as a positive control.

Sequence and Features