Part:BBa_K4601204
NpHR P240T F250Y expression cassette in the Evolution.T7 mutational region
This part is an expression cassette of the NpHR P240T F250Y mutant halorhodopsin (BBa_K4601004).
Usage and Biology
Halorhodopsins (HR) are the second class (after channelrhodopsins) 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, several were already used in preliminary genetic therapeutic tests in animals.
NpHR is a halorhodopsin identified in the archaea Natronomonas pharaonis (Uniprot P15647). Its a mutant P240T, F250Y is able to interact with NpHtrII, while the wild type protein (BBa_K4601003) is not [17].
Opsins’ expression in E. coli and characterisation of their spectral properties
Design
Apart from NpHR, we studied in parallel several other microbial opsins that have also been reported to have potential in vision restoration: CatCh, ChrimsonR, and Jaws, but also NpSR. 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 [4–6] 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
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 T7CGG 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 [7]. This would be vital to concentrate the expressed opsins in the membrane and facilitate their spectroscopic analysis after membrane purification [8].
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 | [9] |
| ChrimsonR | 590 nm | [10] |
| Jaws | 632 nm | [3] |
| NpHR | 589 nm | [11] |
| NpSRII | Around 500 nm | [12] |
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 [8]. 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 [8]. 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 [13]. Unlike what is expected [8], 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 [14]. 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 [8]. 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 [15,8,16,14].
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) [8]. 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).
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] 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.
[5] 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.
[6] 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.
[7] 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.
[8] Maresca JA, Keffer JL, Miller KJ. Biochemical analysis of microbial rhodopsins. Current Protocols in Microbiology (2016) 41: 1F.4.1-1F.4.18.
[9] 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.
[10] 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.
[11] Scharf B, Engelhard M. Blue halorhodopsin from Natronobacterium pharaonis: wavelength regulation by anions. Biochemistry (1994) 33: 6387–6393.
[12] 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.
[13] Keffer JL, Hahn MW, Maresca JA. Characterization of an unconventional rhodopsin from the freshwater actinobacterium Rhodoluna lacicola. Journal of Bacteriology (2015) 197: 2704–2712.
[14] 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.
[15] 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.
[16] 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.
[17] 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1185
- 1000COMPATIBLE WITH RFC[1000]
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