Part:BBa_K4601201

ChrimsonR expression cassette in the Evolution.T7 mutational region

This part is an expression cassette of the ChrimsonR channelrhodopsin (BBa_K4601001).

Usage and Biology

Channelrhodopsins (CR) are the main class of microbial opsins used in optogenetic therapies for visual restoration [1]. They are cation pumps activated by light and have a depolarizing effect [2,3]. Among CR, several were already used in preliminary genetic therapeutic tests in animals and even humans.

ChrimsonR (BBa_K4601001) is a channelrhodopsin derived from the CR Chrimson of Chlamydomonas noctigama that is naturally red-shifted [4]. Compared to the Chrimson, ChrimsonR contains one point mutation K176R that does not modify the absorption properties, but increases the photocurrent generated upon light irradiation. ChrimsonR is a Na⁺ inward pump that is also capable of transporting H⁺, K⁺ and Ca²⁺. It is one of the few microbial opsins in clinical trials for gene therapy for patients with documented diagnosis of non-syndromic Retinitis Pigmentosa [5].

Opsins’ expression in E. coli and characterisation of their spectral properties

Design

Apart from ChrimsonR, we studied in parallel several other microbial opsins that have also been reported to have potential in vision restoration: CatCh, Jaws and NpHR, but also NpSRII. Our plan is to use these proteins as a control to compare them, in terms of light sensitivity and wavelength tuning of their absorption spectrum, with their mutated variants obtained by the Evolution.T7 system and to finally select the variants that show enhanced features with favorable mutations.

To allow the expression of the five opsins, 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 1). For this:

- We designed specific RBS libraries for each opsin using the De Novo DNA’s Library Calculator v2.0 [6–8] 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 T7_CGG 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 T7RNAP_CGG-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

In total we designed six cassettes for the expression of CatCh (BBa_K4601200), ChrimsonR (BBa_K4601201), Jaws (BBa_K4601202), NpHR (BBa_K4601203), NpHR P240T F250Y (BBa_K4601204) and NpSRⅡ (BBa_K4601205).

Figure 1. The general architecture of the Evolution.T7 system. The gene to be evolved is placed under the control of T7 promoter and followed by the T7_CGG promoter in the reverse orientation, flanked upstream and downstream by four T7 terminators.

For this system, the E. coli C43(DE3) strain was chosen for opsin expression as it is a BL21(DE3) derivative, selected for its increased capacity of expressing membrane proteins [9]. This would be vital to concentrate the expressed opsins in the membrane and facilitate their spectroscopic analysis after membrane purification [10].

Knowing that different opsins have different absorption properties (Table 1), we expect to detect a peak in the absorption spectra of the membrane preparations characteristic to the opsin.

Table 1. The different opsins used in this study and their maximum absorption wavelength reported in the literature.
Opsin	Maximum absorption wavelength	Reference
CatCh	480 nm	[11]
ChrimsonR	590 nm	[4]
Jaws	632 nm	[12]
NpHR	589 nm	[13]
NpSRII	Around 500 nm	[14]

Build

The DNA sequences of all opsins mentioned above were synthesized except that of NpSRⅡ which was amplified directly from N. pharaonis genome with primers that introduce specific type IIS restriction sites (BsaI). The pSEVA721 backbone was also amplified by PCR to make it Golden Gate compatible. All of the expression cassettes (BBa_K4601200, BBa_K4601201, BBa_K4601202, BBa_K4601203, BBa_K4601204, BBa_K4601205) were assembled by Golden Gate in the low copy-number plasmid pSEVA721.

Test

Membranes from E. coli C43(DE3) cells expressing the opsin genes were extracted following a protocol adapted from [10]. Briefly, E. coli cells were first grown overnight at 37 °C at 200 rpm in LB (Lennox) supplemented with 10 µg/mL trimethoprim. The cells were then diluted by 100 times in 50 mL of the same media and, after 4 hours of incubation at 37°C at 200 rpm, the opsin expression was induced with 1 mM IPTG and 10 µg/mL all-trans-retinal. As all E. coli DE3 strains, the C43(DE3) contains inserted in its genome the T7 RNA polymerase gene required for expression from the T7 promoter that controls the opsin gene in the Evolution.T7 system. It is under the control of the lacUV5 promoter and requires IPTG to induce expression. Microbial opsins are type I opsins that require all-trans-retinal as co-factor for their activity. This compound is not naturally produced by E. coli, and for this reason we supplement it during culturing.

After an overnight incubation at 37°C at 200 rpm, cells were harvested by centrifugation (3000 g for 15 minutes at 4°C), washed twice in 20 mL of water and submitted to an osmotic lysis in 5 mL “Sweet” buffer containing 0.4 M sucrose, 75 mM TrisHCl pH 8.0 and 2 mM MgSO4₄. After an hour of incubation at 37 °C at 200 rpm, the “Sweet” buffer was removed by centrifugation (3000 g for 15 minutes at 4°C) and the cells resuspended in a “Salt” buffer (0.8 M NaCl, 50 mM Tris pH 7.6, 10 mM MgSO₄). Cells were broken with 1 g of glass beads (1 mm diameter) by vortexing 5 times 1 minute at maximum speed interrupted by 1 minute on ice. The mix was transferred into microcentrifuge tubes and the membrane fraction was collected by centrifugation (15000 g for 20 minutes at 4°C). After discarding the supernatant, the pellet (Figure 2) was solubilised using three different detergents, n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltoside, or sodium cholate, each at a concentration of 3% in 10 mM PIPES-KOH pH 7 buffer in a total volume of 400 µL. After an overnight incubation at 4°C at 1400 rpm, the debris were removed by centrifugation (14000 g for 10 minutes at 4°C) and the supernatant collected.

Absorption spectra were recorded in an opaque wall 96-well polystyrene microplate (COSTAR 96, Corning) using a CLARIOstar (BMGLabtech) plate reader (Figure 4).

SDS-PAGE analysis (Figure 3) was performed on 5 µL of solubilised membranes that were mixed with Laemmli Buffer (final concentrations 20.83 mM Tris-HCl pH 6.8, 0.67% (w/v) SDS, 3.33% glycerol, 1.67% 2-mercaptoéthanol, 0.5% bromophenol blue) and after 1 hour of incubation at room temperature they were loaded onto a 12 % SDS-polyacrylamide gel for protein separation, using a Bio-Rad Protean mini-gel system. Electrophoresis was performed in the SDS-PAGE running buffer (3.03 g/L Tris base, 14.4 g/L Glycine, 1 g/L SDS, pH 8.3) at constant 150 V, until the dye migrated close to the bottom of the gel. The gel was then stained with Coomassie Blue R-250.

Learn

In order to assess the spectral properties of the different opsins, we need to purify the membrane fraction of E. coli C43(DE3) cells that should contain the expressed opsin proteins, according to a protocol adapted from [10]. Such an approach is reported to increase the signal due to the light absorption by opsins and reduce scattering. Moreover, it avoids the use of protein tags that might disrupt the assembly of opsin multimers in the membrane.

By looking at the color with the eye (Figure 2), all the pellets of the different opsins seem to be colorless similar to the negative control in which opsins are absent (First picture on the left), except that of ChrimsonR that shows a slight orange color. The slight orange color could be attributed to the expression of the opsins, but it is important to note that in addition to the expressed opsins, lipids, carotenoids and other colored membrane proteins can be present in the pellet [15]. Unlike what is expected [10], we did not observe a pellet and a darker film on top of it. Instead, we only saw a homogenous pellet. This might be as a result of low expression levels of the opsins due to the use of pSEVA721 low copy number plasmid [16]. As a consequence, we proceeded to the solubilisation of the entire pellet using first the detergent n-octyl-β-D-glucopyranoside, then, in the follow-up experiments, also n-dodecyl-β-D-maltoside and sodium cholate.

Figure 2. E. coli C43(DE3) cell pellets expressing the various opsins (BBa_K4601200, BBa_K4601201, BBa_K4601202, BBa_K4601203, BBa_K4601204, BBa_K4601205), along with the negative control (the pSEVA721 vector).

In order to assess the protein expression, SDS-PAGE was performed for the solubilized membranes extracted from E. coli C43(DE3) cell pellets with the three different detergents (n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltoside and sodium cholate respectively). In order to visualize all of the proteins extracted, the gel was stained with the Coomassie blue dye (Figure 3). The visualization of these proteins of different expression cassettes pellets shows similar patterns as that of the “No opsin” control in the case of each detergent used, suggesting that there is a similar level of membrane proteins expressed between the opsins/no opsins situations, which is logical as we are using the same strain and expression architecture. However, by looking at the gels we can't be certain that our opsins of interest are being expressed, although, we can see some bands with sizes similar to what is expected to obtain from our expression systems (CatCh: 34.31 kDa, ChrimsonR: 39.13 kDa, Jaws: 29.03 kDa, NpHR: 31.08 kDa and NpSR: 23.93 kDa). Nonetheless, we can not neglect the possibility that these bands might correspond to other membrane proteins.

Figure 3. Coomassie blue-stained SDS-PAGE profiles of solubilised membranes extracted from E. coli C43(DE3) cell pellets expressing the various opsins (BBa_K4601200, BBa_K4601201, BBa_K4601202, BBa_K4601203, BBa_K4601204, BBa_K4601205), along with the negative control (the pSEVA721 vector). Solubilisation was performed with three different detergents (n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltoside, sodium cholate).

To check the profile of light absorbance by the different opsins, we measured the absorption of the extracted membrane fractions (first with n-octyl-β-D-glucopyranoside as a detergent) from E. coli C43(DE3) carrying the opsins genes, within the range of visible light wavelengths (300 - 700 nm) (Figure 4). All the spectra obtained in this case (top left) show a peak at 412 nm which we consider as a reference peak as it is the most significant peak in the spectra of the different membranes [10]. Moreover, after normalizing the absorption data of the different membranes with “opsins to that of “no opsins, we can spot a peak at around 560 nm in the case of Jaws, NpHR, NpHR-P240T-F250Y and NpSRII, while this peak did not exist in case of ChrimsonR and Catch. This peak at around 560 nm can be as a result of the absorption by opsins. To investigate if the absence of the peak at position around 560 nm in case of ChrimsoR and CatCh is due to the influence of the detergent used, we repeated the experiment and replaced n-octyl-β-D-glucopyranoside with two other detergents (n-dodecyl-β-D-maltoside or sodium cholate). After subtracting the absorption values of “no opsins” spectra, we noticed slight peaks at around 560 nm for both CatCh and ChrimsonR when n-dodecyl-β-D-maltoside was used. It is obvious that with different detergents the shape of the spectra have changed including the peaks at 412 nm and 560 nm. As a result, this supports that the choice of the detergent has a significant impact on the absorption property.

Not noticing a clear and prevalent peak for the maximum absorbance of the various opsins at their respective wavelengths, as mentioned in Table 1, might be due to various factors such as; type of buffers (affect the pH which in turns alter the absorption) and/or detergents (can cause opsins to bleach) used for membranes purifications, low molar absorptivity (extinction coefficients) of opsins, interference of other light absorbing molecules in the membrane, and the possible low expression of opsins due to utilization of low copy number plasmid pSEVA721 [17,10,18,16].

In conclusion, the results obtained are promising but not sufficiently conclusive to confirm the production of opsins or identify their exact absorbance peaks in the spectra. Therefore, it would be interesting to explore the use of alternative buffers, detergents, and expression vectors to enhance expression capabilities and record the absorption spectra again.

Figure 4. Absorption spectra of membranes extracted from E. coli C43(DE3) carrying the opsins genes under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601200, BBa_K4601201, BBa_K4601202, BBa_K4601203, BBa_K4601204, BBa_K4601205). Solubilisation was performed with three different detergents (n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltoside, sodium cholate). Absorbance values were normalized by the peak at 412 nm (top row) [10]. As a negative control, membranes from E. coli cells carrying an empty pSEVA721 vector were treated alike and the obtained spectra subtracted from the spectra of the opsin containing solubilised membranes (bottom row).

Channelrhodopsins screening systems

To establish a screening system to detect channelrhodopsins activity, we decided to focus our attention on the main cation they transport, which is the Na⁺ and implement in E. coli a Na⁺ biosensor who’s output would be connected to the light activation of opsins. Two different systems schematised in Figure 5 were conceived, tested and described here-after. The first one is based on a Na⁺ riboswitch and the second one on the fluorescent dye CoroNa™ Green.

Figure 5. Design of the channelrhodopsin activity detection systems based on either a Na⁺ riboswitch or on the fluorescent dye CoroNa™ Green.

The Na⁺ riboswitch screening system could not be adapted to E. coli for reasons described on the BBa_K4601021 and BBa_K4601022 pages.

CoroNa™ Green-based detection of channelrhodopsins’ activity

CoroNa™ Green is a chemical compound developed by the company Molecular Probes to be used as a Na⁺ indicator. According to its supplier, “The CoroNa™ Green dye is an improved green-fluorescent sodium (Na⁺) indicator that exhibits an increase in fluorescence emission intensity upon binding Na⁺, with little shift in wavelength”.

For these reasons, we choose to use CoroNa™ Green to evaluate the activity of channelrhodopsins (CR), some of which are light-driven Na⁺ pumps importing sodium ions inside the cell.

Design

CoroNa™ Green dye is commercially available in two versions: one which is cell-permeable and another one which is not. The cell-impermeable version is the one capable of binding the Na⁺ ions and emitting a fluorescence signal, but it cannot penetrate cells which make this version not suitable for our goal of detecting the channelrhodopsin’s activity by evaluating the intracellular Na⁺ content. Moreover, the cell-impermeable version may lead to an increased background due to detection of extracellular Na⁺ ions.

In contrast, the cell-permeable version of CoroNa™ Green dye does not have the same capacity of binding the Na⁺ ions. However, once inside the cell, it can be hydrolysed by cellular esterases and converted into the impermeable version. Thus, fluorescence will be emitted only by intracellular CoroNa™ Green molecules activated by intracellular Na⁺ ions. Release in the environment will only happen upon cell death, which is prevented in the staining protocol by using E. coli growth media throughout the procedure.

CoroNa™ Green is mainly used in the literature in eukaryotic cells and the supplier was unable to provide us with guidance for using it in bacteria. Nevertheless, we found a few publications, all from the same lab, that used CoroNa™ Green in prokaryotes [19,20].

Build

No special genetic constructs were necessary for these tests. We used the above described plasmids expressing the various opsin genes under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601200, BBa_K4601201, BBa_K4601202, BBa_K4601203, BBa_K4601204, BBa_K4601205).

Test

The CoroNa™ Green staining was performed according to a protocol adapted from the one described by Morimoto et al. [20]. Briefly, E. coli C43(DE3) cells were grown overnight at 37 °C at 200 rpm in 12 mL tubes with 3 mL of LB (Lennox) supplemented with 10 µg/mL trimethoprim. The cells were then diluted by 100 times in 20 mL of the same media and after 4 hours of incubation at 37°C at 200 rpm, they were induced with 1 mM IPTG and 10 µg/mL all-trans-retinal. The culture was then split into two and one tube was incubated overnight at 37°C at 200 rpm in dark (covered in aluminum foil) while the other was placed in a shaking incubator equipped with LEDs producing white light at 37°C at 200 rpm. After these overnight incubations, 400 µL of cells were harvested by centrifugation (6000 g for 2 minutes), washed twice in 1 mL of T-broth (1% Bacto tryptone, 10 mM potassium phosphate, pH 7.0) and resuspended in 100 μl of T-broth containing 40 μM CoroNa™ Green (cell permeant version, Molecular Probes C36676) and 10 mM EDTA-KOH pH 8.0. Tubes were covered in aluminum foil (to keep them in the dark) and after one hour of incubation at room temperature in a tube rotator at 5 rpm, cells were harvested by centrifugation (6000 g for 2 minutes) and washed three times in 1 mL of minimal salts (MS) media composed of 50 mM K₂HPO₄, 20 mM NH₄Cl, 4 mM Citric acid, 1 mM MgSO₄, 0.2% glucose, nitrilotriacetic acid 0.01 µM, CaCl₂ 3 µM, FeCl₃ 3 µM, MnCl₂ 1 µM ZnCl₂ 0.3 µM, H₃BO₃ 0.3 µM, CrCl₃ 0.3 µM, CoCl₂ 0.3 µM, CuCl₂ 0.3 µM, NiCl₂ 0.3 µM, Na₂MoO4 0.3 µM, Na₂SeO3 0.3 µM pH 7.2. Finally, cells were resuspended in 200 μl MS media and the suspension transferred in an opaque wall 96-well polystryrene microplate (COSTAR 96, Corning). The CoroNa™ Green fluorescence (λ_excitation 488 nm and λ_emission 530 nm) and optical density at 600 nm (OD600) were measured in a CLARIOstar (BMGLabtech) plate reader before and after the addition of NaCl at a concentration of 100 mM. Fluorescence values were normalized by OD600.

Learn

The CoroNa™ Green staining tests were performed in E. coli C43(DE3) carrying cells the various opsins genes under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601200, BBa_K4601201, BBa_K4601202, BBa_K4601203, BBa_K4601204, BBa_K4601205) or as control an empty backbone (pSEVA721). The opsins’ expression was induced with IPTG and all-trans-retinal was supplemented into the growth media to allow the formation of a functional protein. Cells were grown in the light or, as control, in the dark, followed by CoroNa™ Green staining.

The results presented in Figure 6, show an increased fluorescent output for E. coli cells expressing the ChrimsonR channelrhodopsin when cells were cultured in the light compared to when they were kept in the dark, the dark values being in the same range of values obtained with the empty backbone as negative control. These results provide clear evidence of the light-dependent Na⁺ import by cells expressing ChrimsonR. ChrimsonR is the only opsin for which this experiment showed Na⁺ import activity, which is not unexpected. Indeed, Jaws, NpHR and NpHR P240T, F250Y are halorhodopsins that transport Cl^- ions into the cell, while NpSRII is a sensory rhodopsin that does not have an ion transport activity. For these 4 proteins, the obtained results are as expected: not different compared to the negative control. However, for CatCh we were expecting to obtain some fluorescence output, as this channelrhodopsin is also capable of importing Na⁺ ions. Our results indicate that either CatCh was not significantly expressed or that its activity is too low to be detected by this method.

Figure 6. CoroNa™ Green staining of E. coli C43(DE3) cells carrying the opsins genes under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601200, BBa_K4601201, BBa_K4601202, BBa_K4601203, BBa_K4601204, BBa_K4601205). As a negative control, E. coli cells carrying an empty pSEVA721 vector were treated alike. Fluorescence values were normalized by OD600.

Evolution of ChrimsonR

Having established that the CoroNa™ Green staining is a good tool for detecting a light-dependent increase in the sodium ions content of a cell carrying the ChrimsonR expression cassette, we moved forward and started the experiments for the evolution of ChrimsonR using Evolution.T7 in vivo directed evolution tool developed by the iGEM Evry Paris-Saclay 2021 team.

Design

Evolution of opsin sequences with the aim to select enhanced variants is the objective of our project. With this goal in mind, all our expressing cassettes were designed from the start to express opsins in a genetic configuration compatible with the Evolution.T7 tool (Figure 1).

For the evolution of ChrimsonR, the MG1655^* Δflu ΔpyrF Δung Δnfi mutagenesis dedicated strain was transformed with the plasmid carrying the ChrimsonR expression cassette and an equimolar mix of all BD-T7RNAP and BD-T7RNAP_CGG-R12-KIRV mutagenic plasmids of the Evolution.T7. This choice was made in order to increase the chances of obtaining enhanced variants. Indeed, in 2021, the iGEM Evry Paris-Saclay team established that some base deaminases are more potent then others. While one may be tempted to use the strongest mutator domains, a too high mutagenic rate may quickly lead only to inactive variants. For this reason, we decided to use only the forward or only the reverse mutators : BD-T7RNAP or BD-T7RNAP_CGG-R12-KIRV, respectively. This strategy also limits the burden imposed on the cell by the presence of three plasmids and eliminates the risk of collision between the two polymerases moving in opposite directions on the DNA.

After the in vivo generation of ChrimsonR mutants with the Evolution.T7, cells were stained with CoroNa™ Green as before, and sorted using Fluorescence-activated cell sorting (FACS) (MACSQuant^® Tyto^® Cell Sorter), a technique that allows isolation of cells having the desired fluorescence intensity.

In our case, we expect that the enhanced ChrimsonR variants would be able to have an increased activity in low light intensity conditions compared to the parental protein, thus importing more Na⁺ ions and, as a consequence, display increased fluorescence upon CoroNa™ Green staining.

Build

No special genetic constructs were necessary for the tests. We used the above described plasmid expressing the ChrimsonR gene under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601201).

The Evolution.T7 mutagenic plasmids were kindly provided by the iGEM Evry Paris-Saclay 2021 team.

Test

E. coli MG1655^* Δflu ΔpyrF Δung Δnfi were first co-transformed with the plasmid expressing the ChrimsonR gene under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601201) and with a mix of all the BD-T7RNAP and BD-T7RNAP_CGG-R12-KIRV mutagenic plasmids of the Evolution.T7 in pSEVA221 and pSEVA471 backbones respectively. Control transformations were performed with the plasmids expressing the T7RNAP and the T7RNAP_CGG-R12-KIRV non mutagenic plasmids in pSEVA221 and pSEVA471 empty backbones.

Transformed cells were selected on LB media containing 5 µg/mL trimethoprim, 12.5 µg/mL kanamycin and 25 µg/mL spectinomycin, then grown overnight at 37 °C at 200 rpm in 12 mL tubes with 3 mL of LB supplemented with the same antibiotics. The cells were then diluted by 100 times in 20 mL of the same media and after 4 hours of incubation at 37°C at 200 rpm, they were induced with 200 ng/µL anhydrotetracycline, 1.5 mM L-arabinose and 10 µg/mL all-trans-retinal. The culture was then split into two and one tube was incubated overnight at 37°C at 200 rpm in dark (covered in aluminum foil) while the other was placed in a shaking incubator equipped with LEDs producing white light at 37°C at 200 rpm. After these overnight incubations, 400 µL of cells were stained with CoroNa™ Green as described above.

Flow cytometry analysis was performed on 50 µL of the cell suspension diluted 100 fold in MS media (composition indicated above) supplemented or not with 100 mM NaCl using the MACSQuant^® Analyzer 16 (Miltenyi Biotec). FACS experiments were performed on 750 µL of the cell suspension diluted 100 fold in MS media supplemented with 100 mM NaCl using the MACSQuant^® Tyto^® cell sorter (Miltenyi Biotec) using a single laser operating at 488 nm for excitation and two bandpass filters PE-H 585/40 nm and FITC-H 525/50 nm. The selection was triggered by fluorescence intensity.

Learn

Prior to FACS, a flow cytometry analysis was performed in order to analyze at single cell level the cell suspensions of ChrimsonR expressing the variants evolved in different conditions of presence or absence of the different Evolution.T7 mutagenic plasmids. Bacteria were identified by their size and granularity based on the side scatter (SSC) and the forward scatter (SSC) values as illustrated in Figure 7 A & C, then the fluorescent cells were counted based on the B1(FITC) values (Figure 7 B & D).

The results of these quantifications are presented in Figure 8. The higher number of fluorescent cells were observed in the positive control containing the wild type ChrimsonR protein expressed by the T7RNAP_CGG-R12-KIRV, followed by the population of variants obtained in the corresponding conditions with the BD-T7RNAP_CGG-R12-KIRV mutagenic plasmids. The T7RNAP_CGG-R12-KIRV is the mutant T7RNAP specific to the mutant T7_CGG promoter that is placed in reverse orientation compared to the ChrimsonR gene. However, it retains partially its capacity to recognise the wild type T7 promoter [21] and this can explain the expression of ChrimsonR. We also observe that the wild type T7RNAP is not efficient to drive the expression of ChrimsonR, the number of fluorescent cells being low, in the same range as the negative controls. This difference may be attributed to different promoter strengths that control the two RNA polymerases (pTetA for T7RNAP and pBad for T7RNAP_CGG-R12-KIRV), to the efficiency of induction of their expression (by anhydrotetracycline and L-arabinose respectively), but also by the copy number of the plasmids encoding them 1-3 copies / cell for pSEVA221 and 3-5 copies / cell for pSEVA471 [16].

When comparing the light versus dark growing conditions, we observe a higher number of fluorescent cells in the light conditions for the wild type ChrimsonR. These results are in line with what we observed previously when we stained with CoroNa™ Green the C43(DE3) cells expressing various opsins and measured the fluorescence / OD600 values (Figure 6). These results provide clear evidence of the light-dependent Na⁺ import by cells expressing ChrimsonR.

This same trend was not observed upon analysis of the library of ChrimsonR variants produced with the Evolution.T7 tool. A drop in the fluorescent output is nevertheless not unexpected as a mutation is generally detrimental for the protein activity, while gain of function mutants are rare.

Figure 7. Flow cytometry analysis of CoroNa™ Green stained E. coli MG1655^* Δflu ΔpyrF Δung Δnfi cells carrying the ChrimsonR gene under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601201) along with the BD-T7RNAP_CGG-R12-KIRV mutagenic plasmids of the Evolution.T7 pSEVA471 backbone grown either in the light or in the dark.

Figure 8. Flow cytometry analysis of CoroNa™ Green stained E. coli MG1655^* Δflu ΔpyrF Δung Δnfi cells carrying the ChrimsonR gene under the control of T7 promoter in the Evolution.T7 system in the pSEVA721 backbone (BBa_K4601201) along with the BD-T7RNAP and/or BD-T7RNAP_CGG-R12-KIRV mutagenic plasmids of the Evolution.T7 in pSEVA221 and pSEVA471 backbones respectively. Controls were performed with the plasmids expressing the T7RNAP and/or the T7RNAP_CGG-R12-KIRV non mutagenic plasmids in pSEVA221 and pSEVA471 backbones.

In order to find this rare gain of function mutants, fluorescence-activated cell sorting (FACS) was used on the cell suspensions of ChrimsonR mutant libraries having the highest number of intense fluorescent cells (Figure 9 and Table 2).

Cells having a fluorescence above average were isolated and subsequently re-analysed by flow cytometry (Figure 10) and plated on LB agar plates containing 10 µg/mL trimethoprim. Moreover, a fraction of the sorted cells were subject to a second FACS for further enrichment.

As a control, we used an E. coli culture constitutively expressing sfGFP in the pSB3T5 backbone (BBa_K2675056). In this case the percentage of fluorescent cells determined by flow cytometry was very high reaching 99.4%, confirming the accuracy of our experimental setup. It’s worth noting that the ChrimsonR selected cells reached a fluorescence level compared to the one of sfGFP expressing E. coli cells.

Table 2. Fluorescence-activated cell sorting (FACS) of E. coli cells carrying the ChrimsonR variants generated with the Evolution.T7 tool and stained with CoroNa™ Green (A) or, as control, an sfGFP expressing cassette.
	ChrimsonR + BD-T7RNAPCGG-R12-KIRV (dark)	ChrimsonR + BD-T7RNAPCGG-R12-KIRV (light)	ChrimsonR + BD-T7RNAPCGG-R12-KIRV (resorted)	ChrimsonR + BD-T7RNAP + BD-T7RNAPCGG-R12-KIRV (light)	sfGFP
Triggered events	8049000	11020000	707300	7442000	1749000
Gated events	17654	12840	2292	5578916	423900
Sorted events	5931	10850	773	2190	163300
Aborted events	108	733	5	25	8617
Gated / Triggered events (%)	0.22%	0.12%	0.32%	74.97%	24.24%
Sorted / Triggered events (%)	0.07%	0.10%	0.11%	0.03%	9.34%
Sorted / Gated events (%)	33.60%	84.50%	33.73%	0.04%	38.52%

Figure 9. Fluorescence-activated cell sorting (FACS) of E. coli cells carrying the ChrimsonR variants generated with the Evolution.T7 tool and stained with CoroNa™ Green (A) or, as control, an sfGFP expressing cassette (B). Cells having a fluorescence above average (red upper zone) were isolated. PE-H: 585/40 nm bandpass filter, FITC-H: 525/50 nm bandpass filter.

Figure 10. Flow cytometry analysis of CoroNa™ Green stained E. coli MG1655^* Δflu ΔpyrF Δung Δnfi cells sorted out by FACS based on fluorescence intensity.

In the next step, we selected a total of 20 mutants to individually characterize their activity based on CoroNa™ Green staining followed by Fluorescence / OD600 measurements in a plate reader. For this, first the mutant plasmids were transferred in C43(DE3) cells to allow on one hand a better expression of the opsins than in the K12 strains, but also to achieve a better clonal isolation. Indeed, the mutants are carried by the pSEVA721 backbone which has a very low copy number with between 1 and maximum 3 copies per cell [16], still inside a cell selected by FACS, the possibility of having a mix of different plasmids per cell exist.

The results are currently under analysis.

References

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

[2] Deisseroth K, Hegemann P. The form and function of channelrhodopsin. Science (New York, N.Y.) (2017) 357: eaan5544.

[3] Govorunova EG, Sineshchekov OA, Spudich JL. Emerging diversity of channelrhodopsins and their structure-function relationships. Frontiers in Cellular Neuroscience (2021) 15: 800313.

[4] Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK, Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, Constantine-Paton M, Wong GK-S, Boyden ES. Independent optical excitation of distinct neural populations. Nature Methods (2014) 11: 338–346.

[5] GenSight Biologics. A Phase 1/2a, Open-label, non-randomized, dose-escalation study to evaluate the safety and tolerability of GS030 in subjects with Retinitis Pigmentosa. clinicaltrials.gov (2022).

[6] 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.

[7] 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.

[8] 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.

[9] 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.

[10] Maresca JA, Keffer JL, Miller KJ. Biochemical analysis of microbial rhodopsins. Current Protocols in Microbiology (2016) 41: 1F.4.1-1F.4.18.

[11] Chaffiol A, Caplette R, Jaillard C, Brazhnikova E, Desrosiers M, Dubus E, Duhamel L, Macé E, Marre O, Benoit P, Hantraye P, Bemelmans A-P, Bamberg E, Duebel J, Sahel J-A, Picaud S, Dalkara D. A new promoter allows optogenetic vision restoration with enhanced sensitivity in macaque retina. Molecular Therapy: The Journal of the American Society of Gene Therapy (2017) 25: 2546–2560.

[12] 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.

[13] Scharf B, Engelhard M. Blue halorhodopsin from Natronobacterium pharaonis: wavelength regulation by anions. Biochemistry (1994) 33: 6387–6393.

[14] Ren L, Martin CH, Wise KJ, Gillespie NB, Luecke H, Lanyi JK, Spudich JL, Birge RR. Molecular mechanism of spectral tuning in sensory rhodopsin II. Biochemistry (2001) 40: 13906–13914.

[15] Keffer JL, Hahn MW, Maresca JA. Characterization of an unconventional rhodopsin from the freshwater actinobacterium Rhodoluna lacicola. Journal of Bacteriology (2015) 197: 2704–2712.

[16] 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.

[17] Snodderly DM. Reversible and irreversible bleaching of rhodopsin in detergent solutions. Proceedings of the National Academy of Sciences of the United States of America (1967) 57: 1356–1362.

[18] McCarren J, DeLong EF. Proteorhodopsin photosystem gene clusters exhibit co-evolutionary trends and shared ancestry among diverse marine microbial phyla. Environmental Microbiology (2007) 9: 846–858.

[19] Minamino T, Morimoto YV, Hara N, Aldridge PD, Namba K. The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na⁺ for flagellar protein export. PLoS pathogens (2016) 12: e1005495.

[20] Morimoto YV, Namba K, Minamino T. Bacterial intracellular sodium ion measurement using CoroNa Green. Bio-Protocol (2017) 7: e2092.

[21] Meyer AJ, Ellefson JW, Ellington AD. Directed evolution of a panel of orthogonal T7 RNA polymerase variants for in vivo or in vitro synthetic circuitry. ACS synthetic biology (2015) 4: 1070–1076.

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 1364
  
• 1000
  COMPATIBLE WITH RFC[1000]