Part:BBa_K4601208

NpHR-NpHTRII-EnvZ expression cassette in the Evolution.T7 mutational region

This part is an expression cassette of BBa_K4601008 which is a fusion protein between the Natronomonas pharaonis NpHR halorhodopsin (BBa_K4601003), the NpHtrII transmembrane domain and the E. coli EnvZ histidine kinase domain.

Usage and Biology

After channelrhodopsins, the halorhodopsins (HR) are the second class of microbial opsins used in optogenetic therapies for visual restoration [1]. They are chloride pumps activated by light and have a hyperpolarizing effect [2]. Among HR, two were already used in preliminary genetic therapeutic tests in animals: NpHR and Jaws.

Jaws (BBa_K4601002) is a halorhodopsin derived from Haloarcula (Halobacterium) salinarum (strain Shark) and is naturally red-shifted [3]. Compared to the wild-type HR (Halo57) it contains two mutations K200R and W214F that do not modify the absorption properties, but increase the photocurrent generated upon light irradiation.

NpHR (BBa_K4601003, Uniprot P15647) is a halorhodopsin identified in the archaea Natronomonas pharaonis. Apart from NpHR, N. pharaonis encodes a bacteriorhodopsin which pumps protons from the inside to the outside of the cell and two sensory rhodopsins NpSRI and NpSRII (BBa_K4601005). Sensory rhodopsins are not pumps, but photoreceptors that respond to light and transmit the signal into the cell via a signaling cascade. For this, in N. pharaonis, NpSRI and NpSRII interact with the transducer proteins NpHtrI and NpHtrII, respectively [4]. NpHtrI and NpHtrII are homodimeric membrane proteins that have also a cytoplasmic histidine kinase domain responsible for initiating the signaling cascade that mediates chemotaxis. The interaction between the SR and their cognate Htr transducers are highly specific. However, a mutant halorhodopsin (NpHR P240T, F250Y, BBa_K4601004) was identified that is able to interact with NpHtrII [5].

With the above considerations in mind, and getting inspired by the work of the iGEM Tokyo-NoKoGen 2012 team, we decided to establish a halorhodopsin screening system.

Design

Our halorhodopsin screening system uses fusion proteins composed of the halorhodopsin of interest, the NpHtrII transmembrane domain and the E. coli EnvZ histidine kinase domain (Figure 1). This design is based on several considerations:

- The HR is covalently linked to the NpHtrII transmembrane domain (via a short linker sequence) to ensure their colocalization

- The natural histidine kinase domain of the NpHtrII was removed as it does not specifically interact with an E. coli signaling cascade. It was replaced by an endogenous E. coli histidine kinase domain.

- The E. coli EnvZ was chosen as a histidine kinase domain as it is part of the widely known EnvZ-OmpR two component system [6].

Figure 1. Fusion proteins composed of the halorhodopsin of interest, the NpHtrII transmembrane domain and the E. coli EnvZ histidine kinase domain. These fusion proteins were designed to create a halorhodopsin screening system.

In this system (Figure 2), it is expected that in the presence of light the halorhodopsin activation is transmitted via protein-protein interaction to the NpHtrII transmembrane domain, which in turn leads to the autophosphorylation of the EnvZ histidine kinase domain and subsequently the phosphorylation of the OmpR transcription factor. The phosphorylated OmpR dimerises and binds to specific sequences present in the promoter regions of the ompC and ompF genes thus upregulating their expression. Based on this property, we designed several reporters in which the pOmpC promoter controls the expression of sfGFP (BBa_K4601223), LacZ𝛼 (BBa_K4601233), the ampicillin resistance gene AmpR (BBa_K4601243) or chloramphenicol resistance gene CmR (BBa_K4601253).

Opsin activation should lead to the expression of the reporter gene and the detection of either a fluorescent output, a colorimetric one, or the capacity to grow in the presence of ampicillin or chloramphenicol. The latter systems would constitute a good selection tool for the identification of halorhodopsins with improved properties in different conditions of light. To be functional in E. coli this system requires the knockout of the natural EnvZ gene. For this we constructed two E. coli ∆envZ strains as described above on this page in chapter n°3.

Figure 2: Design of the halorhodopsin activity detection system based on fusion proteins and the E. coli EnvZ/OmpR two component system.

To allow the expression of these fusion proteins, but also their evolution using the tool developed by the iGEM Evry Paris-Saclay 2021 team, we designed expression cassettes following the Evolution.T7 architecture (Figure 3). For this:

- We designed specific RBS libraries for each opsin using the De Novo DNA’s Library Calculator v2.0 [7–9] and selected the one predicted to have a Translation Initiation Rate (TIR) of about 10000.

- We placed this RBS+CDS under the control of the T7 promoter upstream and four T7 terminators downstream

- We also inserted the mutant T7CGG promoter in reverse orientation between the end of the opsin’s coding sequence and the first of the four terminators to ensure the occurrence of mutations driven by the mutant T7RNAPCGG-R12-KIRV linked with a base deaminase (BD) moving in reverse orientation. To stop the ChrimsonR four T7 terminators were placed in reverse orientation upstream of the T7 promoter

Thus, five fusion proteins were designed as follows: for the three halorhodopsins used in optogenetic therapies for visual restoration Jaws-NpHTRII-EnvZ (BBa_K4601007), NpHR-NpHTRII-EnvZ (BBa_K4601008) and its mutant NpHR-P240T-F250Y-NpHTRII-EnvZ (BBa_K4601009). As positive control, we designed the NpSRII-NpHTRII-EnvZ (BBa_K4601010) and as negative control the “no opsin” version NpHTRII-EnvZ (BBa_K4601006).

Figure 3. Schematic representation of the Opsin-NpHTRII-EnvZ fusion proteins expressions cassettes in the Evolution.T7 system (BBa_K4601007, BBa_K4601008, BBa_K4601009, BBa_K4601010).

Build

The expression cassettes of the different opsins-NpHtrII-EnvZ fusions (BBa_K4601207, BBa_K4601208, BBa_K4601209, BBa_K4601210) as well as the negative control the “no opsin” version NpHTRII-EnvZ (BBa_K4601206) were assembled by Golden Gate in the pSEVA721 backbone. This backbone was chosen as it has a very low copy-number [10] compatible with the evolution strategy (if a high copy plasmid is employed, many different mutants will coexist in the same cell which will introduce heterogeneity in the selection process).

The different opsin sequences synthesized were PCR amplified with primers allowing the introduction of specific type IIS restriction sites (BsaI). The NpSRII and the NpHtrII sequences were recovered by PCR from the genome of N. pharaonis (kindly provided by our host lab) and the EnvZ histidine kinase domain from the E. coli genome.

In parallel, four reporter plasmids were assembled by Golden Gate in the pSB3T5 backbone. They contain the pOmpC promoter followed by different reporter genes: sfGFP (BBa_K4601223), LacZ𝛼 (BBa_K4601233), the ampicillin resistance gene AmpR (BBa_K4601243) or chloramphenicol resistance gene CmR (BBa_K4601253).

Test

The first functional tests were performed in E. coli C43(DE3) or C43(DE3) ΔenvZ (ApraR) cells that were co-transformed with two plasmids. The first plasmid carries either the different opsins-NpHtrII-EnvZ fusions in the pSEVA721 backbone (BBa_K4601207, BBa_K4601208, BBa_K4601209, BBa_K4601210), or as controls either an empty backbone (pSEVA721) or an NpHtrII-EnvZ fusion without the N-terminal opsin (BBa_K4601206). The second (reporter) plasmid, on the pSB3T5 backbone, contains the pOmpC promoter followed by different reporter genes: sfGFP (BBa_K4601223), LacZ𝛼 (BBa_K4601233), the ampicillin resistance gene AmpR (BBa_K4601243) or chloramphenicol resistance gene CmR (BBa_K4601253).

Cells were then grown overnight at 37 °C at 200 rpm in 96-deep-well plates with 1 mL of LB (Lennox) supplemented with 5 µg/mL tetracycline and 5 µg/mL trimethoprim. The cells were then diluted by 40 times in the same media and after 4 hours of incubation at 37°C at 200 rpm, they were further diluted by 20 times in media containing also 10 µg/mL all-trans-retinal and 1 mM IPTG in three opaque wall 96-well polystryrene microplates (COSTAR 96, Corning). One plate was incubated at 37°C at 200 rpm and the sfGFP fluorescence (λ_excitation 488 nm and λ_emission 530 nm) and optical density at 600 nm (OD600) were measured every 10 minutes for 24 hours, in a CLARIOstar (BMGLabtech) plate reader. The second plate was incubated at 37°C at 200 rpm in dark (covered in aluminum foil) and a third plate also at 37°C at 200 rpm but in a shaking incubator equipped with LEDs producing white light with an intensity of 2000 lux.

When the reporter gene was sfGFP, after the overnight culture, the fluorescence and the OD600 were recorded with the CLARIOstar plate reader. Fluorescence values were normalized by OD600.

When the reporter gene was LacZ𝛼, after the overnight culture, a Miller assay was performed following the single-step method for β-galactosidase assays in E. coli using a 96-well plate reader [11,12]. For this 80 µL of the overnight cultures were mixed with 120 µL of β-gal mix containing 60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM 2-mercaptoethanol, 0.2 mg/mL lysozyme, 1.1 mg/mL o-nitrophenyl-β-D-galactoside (ONPG), 6.7% PopCultures Reagent pH 7.0. The plate was incubated at 37°C at 200 rpm and the optical densities at 600 nm (OD600) and 420 nm (OD420) were measured every 2 minutes for 2 hours, in a CLARIOstar (BMGLabtech) plate reader with the path correction option turned on. The slope of the variation of OD420 over time (OD420/min) was determined and to calculate the Miller Units, it was multiplied by 1000 and divided by the OD600 and the culture volume used in the assay (80 µL).

When the reporter gene was AmpR or CmR, after the overnight culture, the cell suspension was diluted 100 fold in LB media and 5 µL of the diluted and non diluted culture were deposited on LB agar plates containing 100 µg/mL ampicillin or 35 µg/mL of chloramphenicol respectively. As control, 5 µL of the diluted and non diluted culture were also deposited on plates containing 5 µg/mL tetracycline and 5 µg/mL trimethoprim.

Learn

To assess the functionality of our halorhodopsin screening system, we first evaluated the expression of the different reporter genes in E. coli cells expressing the various opsin-NpHtrII-EnvZ fusions (and the corresponding controls). We chose to perform these tests in the C43(DE3) ∆envZ (ApraR) strain for several reasons:

- It has increased capacity of expressing membrane proteins [13], possess the T7RNAP in its genome, required for the expression of our opsin fusion proteins that are placed under the control of the T7 promoter

- It has a knockout of the envZ gene. Thus the pOmpC promoter controlling the expression of our reporter genes is not activated by changes in the medium osmolarity as it happens in wild type E. coli strains

When using sfGFP as a reporter (Figure 4), we observed generally increased Fluorescence/OD600nm values when bacteria were cultivated in the dark compared to the cells grown in the light or in the plate reader. These results are unexpected, and we suspect this is most probably due to bleaching of GFP by the light. They are thus not conclusive as we are unable to assess the effect of light on the opsins’ activation and the subsequent expression of our reporter gene.

Figure 4. In vivo characterization of sfGFP expression driven by the pOmpC promoter (BBa_K4601223) in E. coli C43(DE3) ΔenvZ (ApraR) cells carrying the different opsins-NpHtrII-EnvZ fusions (BBa_K4601207, BBa_K4601208, BBa_K4601209, BBa_K4601210) according to the experimental design depicted in Figure 2. The controls were performed with an empty backbone (pSEVA721) in both E. coli C43(DE3) expressing naturally the EnvZ gene and E. coli C43(DE3) ΔenvZ (ApraR) cells. An additional control was also performed with only an NpHtrII-EnvZ fusion without the N-terminal opsin (BBa_K4601206). Cells were grown either in the light (under white light produced by LEDS), or in the dark (covered in aluminum foil) or in the plate reader. The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

For this reason, we turned to other reporters to eliminate the direct influence of light on the output.

When using LacZ𝛼 as a reporter and the process of in vivo alpha complementation followed by a colorimetric Miller assay, the results presented in Figure 5 show no significant difference between the negative control (the empty backbone) and the cells carrying the various opsins fusions and this regardless of the presence or absence of light. As a positive control of our assay, we also evaluated the β-galactosidase activity using a LacZ𝛼 constitutive expression cassette (BBa_K4601234). The values were higher, but not as high as those obtained with the the C43(DE3) strain expressing the wild type β-galactosidase, indicating sub effective alpha complementation in the C43(DE3) ΔenvZ (ApraR) Δ(lacZ)M15 cells.

Figure 5. Miller assay for the characterization of LacZ𝛼 expression driven by the pOmpC promoter (BBa_K4601233) in E. coli C43(DE3) ΔenvZ (ApraR) Δ(lacZ)M15 cells carrying the different opsins-NpHtrII-EnvZ fusions (BBa_K4601207, BBa_K4601208, BBa_K4601209, BBa_K4601210) according to the experimental design depicted in Figure 2. The controls were performed with an empty backbone (pSEVA721) in both E. coli C43(DE3) expressing naturally the EnvZ gene and E. coli C43(DE3) ΔenvZ (ApraR) cells. Additional controls were also performed with only an NpHtrII-EnvZ fusion without the N-terminal opsin (BBa_K4601206) and with a LacZ𝛼 constitutive expression cassette (BBa_K4601234). The data and error bars are the mean and standard deviation of at least three measurements on independent biological replicates.

When using AmpR or CmR as reporters and evaluated the growth of E. coli cells in the presence or absence of the corresponding antibiotic, we again observed no difference in the behavior of the different constructs. As shown in Figure 6, all constructs, including the positive and negative controls were able to grow. A leaky expression, even at very low levels, of the pOmpC promoter could lead to the expression of the AmpR and CmR resistance genes. As both the resistance mechanisms involve modification of the antibiotic (and thus its inactivation) by the enzyme encoded by the resistance gene, even a low expression could lead to the appearance of a resistance phenotype corresponding to the antibiotic. As control, we also tested the growth of E. coli cells carrying other reporters under the control of the pOmpR promoter. No growth was observed in these cases (data not shown).

Figure 6. In vivo characterization of the growth of E. coli cells carrying the chloramphenicol acetyltransferase gene (CmR) under the control of the pOmpC promoter (BBa_K4601207, BBa_K4601208, BBa_K4601209, BBa_K4601210) in the presence of 35 µg/mL chloramphenicol, according to the experimental design depicted in Figure 2. The controls were performed with an empty backbone (pSEVA721) in both E. coli C43(DE3) expressing naturally the EnvZ gene and E. coli C43(DE3) ΔenvZ (ApraR) cells. An additional control was also performed with only an NpHtrII-EnvZ fusion without the N-terminal opsin (BBa_K4601206). Cells were plated on LB agar containing 35 µg/mL chloramphenicol.

Based on these four experiments, and notably on the alpha complementation assay, we conclude that the halorhodopsin screening system is not functional in E. coli as such. This may be due to the low expression of the fusion proteins (as described above in chapter n°4 also for the opsins expression alone), most probably due to the very low copy number of the backbone. The RBS strength may also be responsible and increasing it is an alternative for boosting the expression.

In addition, these preliminary tests allowed us to eliminate sfGFP, AmpR, and CmR systems from the list of potential selection markers of opsin activity because of light sensitivity (as in the case of sfGFP) or because their leaky expression is enough to lead to antibiotic resistance (as in the case or AmpR, and CmR).

References

[1] Sakai D, Tomita H, Maeda A. Optogenetic therapy for visual restoration. International Journal of Molecular Sciences (2022) 23: 15041.

[2] Engelhard C, Chizhov I, Siebert F, Engelhard M. Microbial halorhodopsins: light-driven chloride pumps. Chemical Reviews (2018) 118: 10629–10645.

[3] Chuong AS, Miri ML, Busskamp V, Matthews GAC, Acker LC, Sørensen AT, Young A, Klapoetke NC, Henninger MA, Kodandaramaiah SB, Ogawa M, Ramanlal SB, Bandler RC, Allen BD, Forest CR, Chow BY, Han X, Lin Y, Tye KM, Roska B, Cardin JA, Boyden ES. Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Nature Neuroscience (2014) 17: 1123–1129.

[4] Jung KH, Spudich EN, Trivedi VD, Spudich JL. An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli. Journal of Bacteriology (2001) 183: 6365–6371.

[5] Sudo Y, Yamabi M, Kato S, Hasegawa C, Iwamoto M, Shimono K, Kamo N. Importance of specific hydrogen bonds of archaeal rhodopsins for the binding to the transducer protein. Journal of Molecular Biology (2006) 357: 1274–1282.

[6] Mizuno T, Mizushima S. Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes. Molecular Microbiology (1990) 4: 1077–1082.

[7] Reis AC, Salis HM. An automated model test system for systematic development and improvement of gene expression models. ACS synthetic biology (2020) 9: 3145–3156.

[8] Farasat I, Kushwaha M, Collens J, Easterbrook M, Guido M, Salis HM. Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria. Molecular Systems Biology (2014) 10: 731.

[9] Ng CY, Farasat I, Maranas CD, Salis HM. Rational design of a synthetic Entner-Doudoroff pathway for improved and controllable NADPH regeneration. Metabolic Engineering (2015) 29: 86–96.

[10] Jahn M, Vorpahl C, Hübschmann T, Harms H, Müller S. Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR. Microbial Cell Factories (2016) 15: 211.

[11] Schaefer J, Jovanovic G, Kotta-Loizou I, Buck M. Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader. Analytical Biochemistry (2016) 503: 56–57.

[12] Schaefer J, Jovanovic G, Kotta-Loizou I, Buck M. A data comparison between a traditional and the single-step β-galactosidase assay. Data in Brief (2016) 8: 350–352.

[13] Miroux B, Walker JE. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology (1996) 260: 289–298.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2274
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1301