Part:BBa_K1758300

sfGFP controlled by T7 promoter and arsenic operator

This part consists of the sfGFP gene with a translation enhancing 5'-UTR under the control of the T7 promoter and arsenic operator. In combination with the E. coli arsenic repressor arsR, this part can be used as an arsenic biosensor.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 162
  

Characterizaion in cell-free protein synthesis

We tested this device in a crude E. coli cell extract that had been generated from cells expressing the arsenic repressor. In the results shown below, no clear induction is observable when adding arsenic to the reaction.

Induction of arsenic sensor in vitro. For this experiment, a cell extract that already contained the arsenic repressor was used. Error bars represent the standard deviation of three biological replicates.

As high arsenic concentrations inhibit the performance of the cell-free protein synthesis, we normalized the results to this effect. In the corrected date, we observed an induction when adding arsenic up to a concentration of 1.87 mg/L.

Normalized induction of arsenic sensor in vitro. For this experiment, a cell extract that already contained the arsenic repressor was used. The data were normalized to account for the negative effect of arsenic on cell extract performance. Error bars represent the standard deviation of three biological replicates.

Compared to the in vivo results, the response to arsenic was relatively small and we measured a high background signal. We assume that this is due to the different construct we used in vitro. This construct had been optimized for our CFPS by exchanging the natural promoter for the T7 promoter and exchanging mRFP1 for our optimized sfGFP. However, we assume that the repression in the presence of ArsR was not effective enough to observe a clear induction. The reason is most likely that the distance between the T7 promoter and the arsenic operator was too large. The distance was a result of our cloning strategy and would likely be suitable for E. coli promoters. However, the T7 promoter requires the operator to be very close for an efficient repression (Karig et al. 2012).

In addition, we performed an experiment in which the arsenic repressor was not present in the reaction from the beginning, but was encoded on a second plasmid. The plasmid concentrations we used had been predicted by our model. In accordance with the aforementioned results, we observed no clear repression and addition of arsenic showed no effect.

References

Karig, David K.; Iyer, Sukanya; Simpson, Michael L.; Doktycz, Mitchel J. (2012): Expression optimization and synthetic gene networks in cell-free systems. In Nucleic acids research 40 (8), pp. 3763–3774. DOI: 10.1093/nar/gkr1191.