Part:BBa_K1758323

UTR-sfGFP under control of Copper responsive promoter

Copper induceble promoter with an untranslated region and sfGFP for detection via fluorescence

Usage and Biology

CopAP is the central component in obtaining copper homeostasis, which exports free copper from cytoplasm to the periplasm. This is enabled by copper induced activation of the operon transcription via CueR. The CueR-Cu+ is the DNA-binding transcriptional dual regulator which activates transcription (Yamamoto, Ishihama 2005). In our project this part is essential for the in vitro characterization of our copper sensor. We started characterizing it with this device, but data suggested that we could reach higher fluorescence level using a T7 promoter, which was realized in BBa_K1758325.

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 BsaI.rc site found at 29
  Illegal SapI.rc site found at 179
  

in vivo

Our sensor for copper detection consists of CueR a MerR like activator and the copper specific promoter copAP. The promoter is regulated by CueR, which binds Cu ²⁺ ions. We also used a sfGFP downstream the promoter for detection through a fluorescence signal.

For our copper sensor we used the native operator of cooper homeostasis from E.coli K12. We constructed a part (BBa_K1758324) using the basic genetic structur shown in our biosensors.The operator sequence, which includes the promoter (copAP), is regulated by the activator CueR. In BBa_K1758324 we combined a codon optimized version of cueR (BBa_K1758320) under the control of a constitutive promoter with sfGFP under the control of the corresponding promoter copAP (BBa_K1758321)(figure 1). Through the addition of a 5’ UTR upstream of the sfGFP we optimized the expression of sfGFP and increased fluorescence.

Figure 1: The concept of our in vivo copper sensor (BBa_K1758324), which consists of the activator under the control of a constitutive promoter (BBa_K1758320) and the operator and promoter sequence of the copper inducible promoter. An untranslated region in front of the sfGFP, which is used for detection, enhances its expression (BBa_K1758323).

Figure 2: Time course of the induction of a copper biosensor with sfGFP for different copper concentrations in vivo. The data are measured with BioLector and normalized on OD₆₀₀. Error bars represent the standard deviation of two biological replicates.

Figure 3: Fluorescence levels at three different stages of cultivation. Shown are levels after 60 minutes, 150 minutes and 650 minutes.

We tested our in vivo copper sensor with sfGFP as reporter gene, to test the functionality of the system. Moreover, we tested different copper concentrations. The kinetic of our sensors response to different copper concentrations is shown in figure 4. The first ten hours show a strong increase in fluorescence. After that the increase in fluorescence is slower. 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.

In vivo we could show that the adding different concentrations of copper has effects on the transcription levels of sfGFP.

The shown data suggest that sensing copper with our device is possible even if the detectable concentrations are higher than the desireble sensitivity limits. Therfore we tested the copper sensor in our in vitro transcription translation approach.

in vitro

For the characterization of the copper sensor with CFPS we used parts differing from that we used in vivo characterization. For the in vitro characterization we used a cell extract out of cells which contain the plasmid (BBa_K1758320) (figure 4), so that the resulting extract is enriched with the activator CueR. To this extract we added plasmid-DNA of the copper specific promoter copAP with 5’-UTR-sfGFP under the control of T7-promoter (BBa_K1758325) to the cell extract. The T7-promoter is needed to get a better fluorescence expression.

Figure 4: To produce the cell extract for in vitro characterization a construct (BBa_K1758320) with copper activator under the control of a constitutive promoter and strong RBS (BBa_K608002) is needed.

Figure 5: T7-copAP-UTR-sfGFP BBa_K1758325 used for in vitro characterization.

The results presented in figure 6 illustrate the influences of different copper concentrations on the cell extract.

Figure 6: Influence of different copper concentrations on our crude cell extract. Error bars represent the standard deviation of three biological replicates.

As shown in figure 6 copper has no negative influence on the functionality of our cell extract. Therefore, a relatively stable system for copper sensing is provided. First tests with specific cell extract and different copper concentrations lead to further tests and normalizations, illustrated in figure 7.

Figure 7: Copper specific cell extract made from E. coli cells which have already expressed the activator before cell extract production. Induction of copper inducible promoter without T7 upstream of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates.

Figure 8: Copper specific cell extract made from E. coli cells which have already expressed the activator before cell extract production. Induction of copper inducible promoter without T7 in front of the operator site with different copper concentrations. Error bars represent the standard deviation of three biological replicates. Data are normalized on coppers influence to the cell extract.

In addition,we measured the operator device under the control of T7 promoter as described before.

Fluorescence was normalized to influence of copper on the the cell extract (figure 9 and figure 10).

Figure 9: Copper specific cell extract made from E. coli cells which have already expressed the activator before cell extract production. Induction with different copper concentrations. Error bars represent the standard deviation of three biological replicates.

Figure 10: Copper specific cell extract made from E. coli cells which have already expressed the activator before cell extract production. Induction of copper inducible promoter with different copper concentrations. Error bars represent the standard deviation of three biological replicates. Data are normalized on coppers influence to the cell extract.

Compared to the former fluorescence levels the T7 reporter device showed higher levels. Therefore, a reporter device under the control of T7 promoter is more suitable for our CFPS.

After normalizing on coppers influence to the cell extract these differences were even more obvious.

Refrences

Grass, Gregor; Rensing, Christopher (2001): Genes Involved in Copper Homeostasis in Escherichia coli, checked on 8/26/2015. Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015.

Outten, F. W.; Outten, C. E.; Hale, J.; O'Halloran, T. V. (2000): Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. In The Journal of biological chemistry 275 (40), pp. 31024–31029. DOI: 10.1074/jbc.M006508200.

Yamamoto, Kaneyoshi; Ishihama, Akira (2005): Transcriptional response of Escherichia coli to external copper. In Molecular microbiology 56 (1), pp. 215–227. DOI: 10.1111/j.1365-2958.2005.04532.x.