Difference between revisions of "Part:BBa K1758352"

Latest revision as of 20:02, 20 September 2015

Nickel responsive promoter with UTR-sfGFP

nickel induceble promoter with 5´untranslated region and sfGFP for detection

Usage and Biology

This Sequence is a part of our nickel sensor BBa_K1758353 . Our Nickel biosensor consists of parts of the rcn-operon from E. coli which codes for a nickel- and cobalt-efflux system. This system is highly sensitive to nickel. The nickel responsive promoter rcnA is inhibited through RcnR, which binds in absence of nickel or cobalt to the operator. With Ni(II)-ions present the repression of the promoter rcnA will be reversed, because the repressor RcnR binds nickel-ions and cannot attach to the DNA (EPA et al., 2013; Blaha et al., 2011; Iwig et al., 2006). For our biosensor we construct the part ( BBa_K1758353 by using the basic construction showed in . For this part we used the repressor RcnR under control of a constitutive promoter ( BBa_K1758350 ) and the nickel specific promoter RcnA with a 5’UTR in front of sfGFP ( BBa_K1758352 ) as reporter protein.

Sequence and Features

Results

in vivo

We aimed to construct a sensor for nickel detection. It consists of rcnR the repressor and the nickel specific promoter prcnA. The promoter is regulated by the RcnR, which binds Ni²⁺-ions. As the former sensors this one encloses a sfGFP for detection via fluorescence.

Our nickel biosensor consists of parts of the rcn-operon from E. coli, which encodes a nickel- and cobalt-efflux system. This system is highly sensitive to nickel. In absence of nickel or cobalt RcnR binds to the operator and inhibits the nickel responsive promoter. With Ni²⁺-ions present the repression of the promoter prcnA will be reversed, because the repressor RcnR binds Ni²⁺-ions and cannot attach to the DNA. For our biosensor we construct the part BBa_K1758353 by using the basic construction shown in figure 1. For this part we used the repressor RcnR under control of a constitutive promoter ( BBa_K1758350 ) and the nickel specific promoter PrcnA with a 5’UTR in front of sfGFP ( BBa_K1758352 ) as reporter protein.

We tested our nickel sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover we tested different concentrations. The kinetic of our sensors response to different nickel concentrations is shown in figure 2. The first five hours show a strong decrease in fluorescence. After that there is a slight increase in fluorescence. Starting levels of fluorescence are not reached. For better visualization the kinetics of figure 2 are represented as bars in figure 3. A fluorescence level difference for 60 min, 150 min and 650 min is represented.

The data for our nickel sensor show a trend that differs for that of the other sensors. There is no indication for a working sensor in vivo (Figure 2 and 3). There is a fluorescence signal, but it decreases in the first five hours. After reaching a minimum the fluorescence increases slowly. Additionally, there is no difference in fluorescence as response to various nickel concentration. Nickel could influence the cells and thereby caused a precipitation, which could result in decrease of fluorescence. With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Due to this observation, no in vitro data using CFPS were taken.

To summarize

With this sensor no production of sfGFP via fluorescence level change could be detected. Therefore, this sensor is not suitable for our approach. Consequently, no in vitro tests were performed. To create a working sensor based on this concept further optimization is needed.