Part:BBa_K1758324

Copper activator under control constitutive promoter and strong RBS and Copper responsive promoter

Usage and Biology

For our copper sensor we used the native operator of copper homeostasis from E.coli K12. This includes the promoter copAP and its regulator CueR. CueR is a MerR like regulator, which stimulates the transcription of copA, a P-type ATPase pump (Outten et al. 2000). CopA is the central component in obtaining copper homeostasis, it exports free copper from cytoplasm to the periplasm. This is enable 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) To sum it up CueR regulon plays an important role in aerobic copper tolerance in E.coli (Grass, Rensing 2001).In BBa_K1758324 we combined the codon optimized CueR (BBa_K1758320) under the control of a constitutive promoter with strong RBS(BBa_K608002)with copAP and sfGFP (BBa_K1758321) for measuring output signals. Through the addition of a 5’UTR before the sfGFP we optimized the expression of sfGFP and increased.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 475
  Illegal SapI.rc site found at 625
  

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 showed 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 before 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).

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

Our sensors were cultivated in the BioLector. Due to the accuracy of this device we could measure our sample in duplicates.

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 2. The first 10 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.

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.

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.

=Use by Oxford iGEM 2016= Our project was to investigate a probiotic treatment for the copper-accumulation disorder: Wilson's disease. This required a system able to detect dietary copper, ideally in the range over which copper concentration changes after a meal (around 5-10μM) and produce a copper chelating protein. In the course of the project we investigated the E coli. CueR-linked copper sensing system and started from from part. However it can be seen from the sequence that the part is actually very different from the subparts suggested which suggests that CueR is expressed divergent from the sfGFP (on the opposite strand and transcribed in the opposite direction): However if you look at the sequence level above this is clearly not the case. The constitutive promoter and the CueR start codon are at the 5’ end of the sfGFP coding strand and the CueR stop codon just upstream of pCopA. The part in fact has the constitutive promoter on the same strand as pCopA and sfGFP facing in the same direction and would be better represented like this:

https://static.igem.org/mediawiki/2016/0/09/PCopA_CueR_incorrect_LOL_sam_oxford_2016.png

As the two coding regions are not separated by a transcription terminator, there would be read through from the constitutive promoter to the sfGFP and sfGFP would be expressed even in the absence of copper. As no negative control is included in the plate reader graph they provide and no settings provided for their BioLector experiments in their protocols it is unclear just how high the expression level at 0mM copper was for this part compared to a negative control strain.

The CueR subpart BBa_K1758320 making up BBa_K1758324 is also incorrectly labelled.

We flipped the CueR and the constitutive promoter to face the opposite direction on the opposite strand i.e. so they were actually divergent. We also had to remove the 5'UTR, because it was too AT rich to be synthesised. This formed our part: https://parts.igem.org/Part:BBa_K1980006 which we used to control the expression of our copper chelators