Part:BBa_K4601200

CatCh expression cassette in the Evolution.T7 mutational region

This part is an expression cassette of the CatCh channelrhodopsin (BBa_K4601000).

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.

CatCh is a channelrhodopsin derived from the ChR2 of Chlamydomonas reinhardtii [4], from which it only retains the first 309 amino acids containing the N-terminal transmembrane domain forming. Compared to ChR2, it also contains one mutation L132C that modifies the photocurrent generated upon light irradiation. CatCh is a Ca2+ inward pump that is also capable of transporting, although to a lesser extent, Na⁺, K⁺ and Mg²⁺. It was successfully used in optogenetic therapies for visual restoration in primates [5].

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

Design

Apart from CatCh, we studied in parallel several other microbial opsins that have also been reported to have potential in vision restoration: ChrimsonR, Jaws and NpHR, 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 [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.

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] Kleinlogel S, Feldbauer K, Dempski RE, Fotis H, Wood PG, Bamann C, Bamberg E. Ultra light-sensitive and fast neuronal activation with the Ca²⁺-permeable channelrhodopsin CatCh. Nature Neuroscience (2011) 14: 513–518.

[5] 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.

[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.

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 NgoMIV site found at 924
  Illegal AgeI site found at 809
  Illegal AgeI site found at 1241
  
• 1000
  COMPATIBLE WITH RFC[1000]