Part:BBa_K2120002

araC+pBAD+B0032+mazF

arac-pBAD is an arabinose induced promoter, mazF is a toxin gene from E.coli. MG1655. This part is functioned as a controlable killer device.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Functional Parameters

Test the toxin gene can be translated and play a role in situations

The 2016 BIT-China iGEM Team equipped the bacteria with a plasmid-sensing logically adjustable cell killer(P-SLACKiller). To kill the slacker bacteria in time, we chose to construct circuits with toxin genes mazF and hokD. And here we designed two circuits to verify the function, one was araC+pBAD+B0032+mazF, another was araC+pBAD+B0032+hokD(BBa_K2120003). The araC+pBAD promoter can be controlled tightly by using arabinose. Then we constructed two plasmids containing these two circuits, and transformed the two plasmids into E.coli BMTop10 respectively. After transformation, we measured the OD600 to draw the growth curve and observed the function of toxin protein. Besides the above two bacteria contained toxin genes, the control was the empty pSB1C3 vector. Add arabinose or not, there was another comparison. We add 10% arabinose 50 uL into 50 mL LB culture medium when the OD600 is 0.6 (the log phase). We measured the OD600 every hour until the bacteria reached the stationary phase.

Fig.1 Compared with the negative control (the empty pSB1C3 vector), OD600 of the two circuits containing toxin proteins are obviously lower, and the difference is evident as time going on. No obvious difference observed among the three groups with no induction. It showed that toxin proteins didn’t leak out.

Through growth curves, we concluded that toxin protein MazF and HokD have different lethal efficiency. Depending on different situations, they can be used.

Regulate the lethal efficiency of the toxin gene in an advanced way

To verify the toxin gene in an advanced way, we tested whether the toxin gene could be a well-regulated one. So we adjusted the translation efficiency of toxin proteins through replace ribosome binding site(RBS), thus to regulate the toxin gene. Through one-step mutation, we have separately replaced the B0032 (33.96%)in above circuits with B0031 (12.64%) and B0034 (100%). So we got： araC+pBAD+B0031+mazF (BBa_K2120004)， araC+pBAD+B0031+hokD(BBa_K2120005)， araC+pBAD+B0034+mazF(BBa_K2120006)， araC+pBAD+B0034+hokD(BBa_K1602043)，and here were the sequencing results.

Fig.2 The successful sequencing results of 4 mutations: B0031+mazF; B0034+mazF; B0031+hokD; B0034+hokD

By using the above methods that we used to verify the function of the toxin gene, we also tested OD600 to get the growth curve to show the lethal efficiency of MazF and HokD under different RBS. Compare with the negative control (pSB1C3 empty vector) and the positive control (B0032 circuits, the above two circuits), the experimental groups showed obvious difference. For MazF, the strong RBS B0034 was proved to have highest lethal efficiency. For HokD, the weak RBS B0031 was proved to have the lowest lethal efficiency.

Fig.3 For the toxin protein MazF, we concluded that the influence of strong RBS is more evident.

Fig.4 For the toxin protein HokD, the replacement of RBS will influence the expression of HokD. The weaker RBS B0031 leads to lower expression of toxin protein HokD.

All results showed both the toxin genes can be well-regulated by different RBS, thus the toxin proteins can be widely used in various situations according to different requirements. Meanwhile, arabinose can be the sensible killer switch to induce araC+PBAD promoter.