Part:BBa_K1758320

copper activator under control constitutive promoter and strong RBS

Activator for copper induceble promoter copAP under the control of constitutive promoter (K608002)

Usage and Biology

cueR is a merR like regulator, which stimulates the transcription of copAP, a P-type ATPase pump (Outten et al. 2000). CopAP is the central component in obtaining copper homeostasis, it 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). This part was used for our in vivo and in vitro characterisation. The CueR serve in our systems as activator and regulate the discription of sfGFP.

***See below for information about use of the part by Oxford iGEM in 2016.***

Functional Parameters

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
  COMPATIBLE WITH RFC[1000]

*****Oxford iGEM 2016*****

The annotation above suggests that CueR is expressed from a constitutive promoter on the bottom strand at the the 3' end. Looking at the sequence reveals that the promoter is actually on the top strand at the 5' end. The CueR start codon begins at nucleotide 62 and the stop codon finishes at the 5' end of this part. All composite parts containing this part are also affected.

in vivo characterisation

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.

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

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.

Contribution from Squirrel-Shenzhen 2024

we utilized CueR (BBa_K1758320) and pCoA, an endogenous regulatory module, to construct a dual plasmid system for detecting copper ions in the environment.

We believe that CueR (BBa_K1758320) initiated by different promoters can lead to varying outcomes for the entire system. Promoters are regulatory switches for gene expression, determining under what conditions the downstream genes are activated. We selected several different promoters from the E. coli genome and replaced the original J23119 promoter in the dual-plasmid system to examine the effects these changes would have on the regulatory system.

We picked 96 single colonies from the plate into liquid LB medium for overnight cultivation. The next day, after diluting the overnight culture to logarithmic phase, we added 0 µM and 500 µM copper ion solutions and tested their fluorescence signals overnight.

We observed that all the promoters exhibited induction strength, indicating that each promoter had some level of activation. The lowest showed a 5-fold induction strength, while the highest reached 10-fold.

We tested the sequences of these promoters using Sanger sequencing, matched them to their corresponding strengths, and established a model between the induction strength and the sequence characteristics.

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.

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