Engineering

Overview

CueR family proteins are key metal regulatory proteins in Escherichia coli, specifically responsible for sensing monovalent copper ions (Cu⁺) in both intracellular and extracellular environments. They belong to the MerR family of transcriptional regulators and play an important role in regulating copper ion homeostasis and resistance to copper toxicity. When the external copper ion concentration increases, CueR binds to copper ions with high affinity. Once copper ions enter the cell, CueR rapidly binds to them, triggering a conformational change in the CueR protein[1,2].

In the absence of copper ions, CueR binds to the promoter region (pCoA) in a repressive state, preventing the expression of downstream genes. When CueR binds to copper ions, it undergoes a conformational change, transitioning from a repressive state to an active state, which then initiates the expression of downstream genes. These genes include[3]:

CopA: Encodes a copper ion-transporting ATPase that expels excess copper ions from the cell, maintaining copper ion balance.

CueO: Encodes a multicopper oxidase responsible for oxidizing copper ions, thereby reducing their toxicity.

CueR regulates the expression of these genes, helping the cell to quickly respond and expel excess copper ions in high-copper environments. Leveraging this mechanism, we constructed a dual-plasmid system:

1. The first plasmid contains the transporter protein CueR driven by a strong promoter:
   • J23119-BBa_B0034- CueR - BBa_B1005
   
2. 2. The second plasmid contains a CueR-sensitive pCoA promoter, with a downstream encoded fluorescent protein[4]:
   • CopA -BBa_B0030- mVenusNB - BBa_B1005
   

By testing the fluorescent protein signal, our system can respond to the copper ion concentration in the environment. Our engineering steps included:

1. Validating the response of this dual-plasmid system to different copper ion concentrations.
2. Testing the response of a single plasmid to various copper ion concentrations to discuss the regulatory system design of endogenous proteins.
3. Screening promoters for CueR expression.

Cycle 1


Design

Copper efflux regulator (CueR) is a Cu+- and Ag+- sensing metalloregulator in E.coli that controls the expression of two genes involved in metal homeostasis: CopA, which encodes a copper/silver ATPase, and CueO, which encodes a copper oxidase [5]. Like most other metalloregulators, CueR acts on promoter DNA that exceeds the optimal length (~17bp) for recognition by sigma70, a subunit of RNA polymerase (RNAP) [5,6]. Taking advantage of this endogenous Cu(II) sensor system, we designed a simplified prototype plasmid to measure the concentration of Cu(II) ions.

This reporting plasmid contains a CueR-regulated copper inducible promoter (BBa_K190017) controling downstream fluorescent protein(FP) as reporter protein. When faced with Cu2+ containing environment , the CueR protein in the E.coli will be activated and bind to the corresponding promoter on our reporter plasmid to start the downstream transcription of reporter FP.

Build

We successfully constructed these two plasmids and transformed them into DH5α cells. After overnight cultivation, we diluted the culture to the logarithmic phase the next day and added the corresponding concentrations of copper ions.

Test

We monitored the growth status and fluorescence intensity of the bacterial culture in this expression system at different copper ion concentrations, as shown in the figure. Additionally, after the system reached a steady state, we recorded the final fluorescence values and plotted a response curve against the different copper ion concentrations[7].

Learn

Here we show the kinetic curves at different copper ion concentrations (Fig. 2a), and we can see that the system exhibits a significant difference from the blank group even at a low copper ion concentration of 170nM within 10hrs. We selected the data points at 35hr for fitting the induced response curve, and it can be found that the reporter system is basically saturated at 250uM Cu2+ concentration(Fig. 2b). This work demonstrate that our design to construct a copper biosensor using is feasible, revealing the effects of the CueR of chassis bacteria overlooked by previous work utilizing this promoter.

Cycle 2


Design

Since CueR is an endogenous protein in Escherichia coli, naturally expressed and involved in downstream regulation in the presence of copper ions, we proposed a hypothesis: If we only transform a reporter plasmid containing the pCoA promoter without expressing additional CueR, could the naturally occurring CueR in the E. coli genome activate the expression of the fluorescent protein? This would reduce the need for plasmids, lessen the burden on the engineered bacteria, and potentially simplify the system.

Build

To validate this hypothesis, we transformed the reporter plasmid containing only the pCoA promoter into DH5α, creating a strain without exogenous CueR expression.

Test

We tested the fluorescent protein signal of this E. coli strain containing only one plasmid under different copper ion concentrations, and compared it with the response of the engineered strain containing two plasmids to varying copper ion concentrations.

The results showed that when only the promoter was provided, it could still respond to copper ions normally and activate the expression of the downstream fluorescent protein based on different copper ion concentrations. We plotted the relationship between the maximum fluorescence values and copper ion concentrations, and found that the system also reached its maximum response at 250 μM Cu²⁺.

Learn

At the same time, we compared the response curves of these two different plasmid systems:

Through the experiment, we concluded that our designed single-plasmid system can also generate a fluorescent signal in a copper-rich environment. This system utilizes the endogenous regulatory protein of E.coli, meaning the expression level of *E. coli*'s own regulatory protein is sufficient to activate the additional reporter system. In future designs of regulatory mechanisms, if the regulatory protein is naturally present in *E. coli*, there may be no need to encode this protein on a plasmid for exogenous expression. Instead, we can attempt to directly use the regulatory protein expressed from the genome, thereby reducing the burden on the engineered bacteria.

Upon comparison, we found that the results of the dual-plasmid system were not as strong as those of the single-plasmid system in terms of signal strength. However, the overall curve became steeper, and the induction threshold for low copper ion concentrations was lower. This leads us to speculate that different expression levels of CueR might cause different system responses.

Cycle 3


Design

Based on the above experiments, we believe that CueR 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.
Build

We purchased a promoter library with Golden Gate standard interfaces. After receiving the library, we performed Golden Gate Assembly (GGA) with plasmid 1. After the reaction, we mixed the GGA product with plasmid 2 and co-transformed them into DH5α cells. The transformed cells were plated on antibiotic-selective agar plates and incubated overnight at 37°C, resulting in single colonies, thus completing the library construction.

Test

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. The results for the 0 µM Cu ion (non-induced) condition are shown in Figure 1, and we extracted the maximum values to create a bar chart and a heatmap.

For the condition with 500 µM Cu ions (induced), we similarly extracted the maximum values and created a bar chart and a heatmap:

Finally, we defined the induction strength as:

Induction strength = RFU(500 µM Cu) / RFU(0 µM Cu), and plotted bar charts and heatmaps of the induction strength for the 96 different promoters in the system:

Learn

We observed that all promoters in the promoter library exhibit a certain level of promoter activity, indicating their significant role in transcriptional regulation. The initiation strength of the promoters shows a degree of variability, with the lowest induction strength being 5-fold, and the highest reaching up to 10-fold. To gain a deeper understanding of the relationship between the activity of these promoters and their sequence characteristics, we employed Sanger sequencing to determine the nucleotide sequences of these promoters. Subsequently, based on the sequencing results, we conducted a correlation analysis between the initiation strength of the promoters and their sequence features, and built a mathematical model accordingly. The detailed construction process and parameter settings of this model can be consulted on our provided "Model" page. Through this model, we aim to reveal the specific mechanisms by which promoter sequence characteristics affect their initiation activity, providing a theoretical foundation and experimental guidance for subsequent research on gene expression regulation.

References
[1] Hu, Y. & Liu, B., 2024. The copper efflux regulator (CueR). Subcellular Biochemistry, 104, pp.17-31. doi:10.1007/978-3-031-58843-3_2.

[2] Checa, S.K. & Soncini, F.C., 2011. Bacterial gold sensing and resistance. Biometals, 24(3), pp.419-427. doi:10.1007/s10534-010-9393-2.

[3] Zhou, X., Xiang, Q., Wu, Y., et al., 2024. A low-cost and eco-friendly recombinant protein expression system using copper-containing industrial wastewater. Frontiers in Microbiology, 15, p.1367583. Published on 21 March 2024. doi:10.3389/fmicb.2024.1367583.

[4] Lischik, C.Q., Adelmann, L. & Wittbrodt, J., 2019. Enhanced in vivo-imaging in medaka by optimized anaesthesia, fluorescent protein selection and removal of pigmentation. PLoS One, 14(3), p.e0212956. Published on 7 March 2019. doi:10.1371/journal.pone.0212956.

[5] Brown, N.L., Stoyanov, J.V., Kidd, S.P. & Hobman, J.L., 2003. The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 27(2-3), pp.145-163.

[6] Phillips, C., Canalizo-Hernandez, M., Yidirim, A., Schatz, G.C., Mondragon, A. & O'Halloran, T.V., 2015. Allosteric transcription regulation via changes in the overall topology of the core promoter. Science, 349(6250), pp.877-881.

[7] Wang, Z.K., Gong, J.S., Su, C., et al., 2024. Multilevel systematic optimization to achieve efficient integrated expression of Escherichia coli. ACS Synthetic Biology, 13(9), pp.2887-2898. doi:10.1021/acssynbio.4c00280.