1 00:00:00,260 --> 00:00:11,789 [Music] 2 00:00:17,560 --> 00:00:14,980 after everyone I area from just 3 00:00:19,750 --> 00:00:17,570 University in Prague Czech Republic and 4 00:00:21,970 --> 00:00:19,760 today I'm going to introduce to my 5 00:00:24,070 --> 00:00:21,980 project test of genetic code evolution 6 00:00:26,230 --> 00:00:24,080 hypothesis all the best evolution of the 7 00:00:29,859 --> 00:00:26,240 RNA binding domain of the ribosomal 8 00:00:32,520 --> 00:00:29,869 protein at 11:00 okay first of all I 9 00:00:34,979 --> 00:00:32,530 introduced like how was a ball with a 10 00:00:38,500 --> 00:00:34,989 pathetically was evolved with the 11 00:00:41,799 --> 00:00:38,510 genetic code different theory assumes 12 00:00:44,770 --> 00:00:41,809 that the original genetic code coding 13 00:00:47,829 --> 00:00:44,780 only for a small set of amino acid a 14 00:00:49,979 --> 00:00:47,839 dismal set of amino acid were introduced 15 00:00:52,989 --> 00:00:49,989 like in the answer to prebiotic protein 16 00:00:55,509 --> 00:00:52,999 but how the scientist like the side 17 00:00:58,149 --> 00:00:55,519 which an amino acid I've introduced are 18 00:01:01,000 --> 00:00:58,159 like early one and which other one could 19 00:01:02,289 --> 00:01:01,010 be like late amino acid particularly 20 00:01:05,890 --> 00:01:02,299 this guy Hicks 21 00:01:07,870 --> 00:01:05,900 Audrey's like summarize all the 22 00:01:11,170 --> 00:01:07,880 different theories and also all the 23 00:01:14,020 --> 00:01:11,180 different evidence about the evidence 24 00:01:16,240 --> 00:01:14,030 abundancy of the different amino acid in 25 00:01:18,130 --> 00:01:16,250 for instance in the Miller experiment 26 00:01:21,279 --> 00:01:18,140 Mathieu right or in the presence of 27 00:01:24,160 --> 00:01:21,289 hydrothermal vents so it just classify 28 00:01:27,099 --> 00:01:24,170 all the amino acid amazing of the de 29 00:01:29,590 --> 00:01:27,109 árbol dance and wonders in this in this 30 00:01:33,639 --> 00:01:29,600 environment and here in the table we can 31 00:01:36,490 --> 00:01:33,649 see that in a table we can see we can 32 00:01:38,289 --> 00:01:36,500 split the the chart into part the first 33 00:01:40,510 --> 00:01:38,299 part are represented like the early 34 00:01:44,050 --> 00:01:40,520 amino acid in see the bottom part of the 35 00:01:45,429 --> 00:01:44,060 chart is like the late immunity because 36 00:01:47,740 --> 00:01:45,439 we can also find like some evidence 37 00:01:49,569 --> 00:01:47,750 indirectly in the genetic code in fact 38 00:01:51,279 --> 00:01:49,579 we will see that the in green is 39 00:01:54,489 --> 00:01:51,289 represented like the early immunity the 40 00:01:57,249 --> 00:01:54,499 represent more than 60 percentage of the 41 00:01:59,550 --> 00:01:57,259 coding the code on that coding for amino 42 00:02:02,709 --> 00:01:59,560 acid respect to the late amino acid but 43 00:02:06,160 --> 00:02:02,719 for the moment are just like sampling 44 00:02:08,999 --> 00:02:06,170 just theory so no one and as demonstrate 45 00:02:13,990 --> 00:02:09,009 experimental II this different theories 46 00:02:15,910 --> 00:02:14,000 so my project is to verify that it's 47 00:02:18,940 --> 00:02:15,920 possible that a protein composite of 48 00:02:19,390 --> 00:02:18,950 only early amino acid amino is it is 49 00:02:23,860 --> 00:02:19,400 still 50 00:02:26,380 --> 00:02:23,870 Lentini function or extraction and so 51 00:02:28,270 --> 00:02:26,390 how to do this feel more or less is the 52 00:02:30,039 --> 00:02:28,280 flowchart of my project so the first 53 00:02:33,160 --> 00:02:30,049 step is like the selection of a target 54 00:02:34,990 --> 00:02:33,170 protein like a modern protein with some 55 00:02:37,420 --> 00:02:35,000 faction function could be like an enzyme 56 00:02:40,270 --> 00:02:37,430 or like a binding protein after that 57 00:02:43,420 --> 00:02:40,280 generate the library compose it only of 58 00:02:45,940 --> 00:02:43,430 early amino acid so substitute all the 59 00:02:47,170 --> 00:02:45,950 late amino acid with the early one and 60 00:02:49,539 --> 00:02:47,180 the next step will be the 61 00:02:51,009 --> 00:02:49,549 characterization of the mutant so 62 00:02:53,470 --> 00:02:51,019 understand if the Alpha recently the 63 00:02:56,940 --> 00:02:53,480 mutant maintain the structure maintain 64 00:03:00,339 --> 00:02:56,950 the function or both or nothing 65 00:03:03,580 --> 00:03:00,349 okay so the first step was the target 66 00:03:05,770 --> 00:03:03,590 was the selection of the target to do 67 00:03:08,050 --> 00:03:05,780 this we have to follow some strictly 68 00:03:11,170 --> 00:03:08,060 criteria the first one the protein the 69 00:03:13,420 --> 00:03:11,180 target should be small so between 50 to 70 00:03:16,119 --> 00:03:13,430 100 amino acid this is more related to 71 00:03:17,649 --> 00:03:16,129 the most practical part because of 72 00:03:19,690 --> 00:03:17,659 course if we had like a small protein 73 00:03:22,539 --> 00:03:19,700 more than 100 no is it we will generate 74 00:03:25,509 --> 00:03:22,549 a library of like more than 10 to the 75 00:03:28,180 --> 00:03:25,519 power of 3040 mutants that it's 76 00:03:31,390 --> 00:03:28,190 impossible to use like in practical way 77 00:03:33,490 --> 00:03:31,400 to manage this kind of library second 78 00:03:36,339 --> 00:03:33,500 step the protein should be like conserve 79 00:03:38,020 --> 00:03:36,349 it and also this step this criteria is 80 00:03:40,930 --> 00:03:38,030 really important in order to reduce the 81 00:03:43,960 --> 00:03:40,940 size of library because we'd like we can 82 00:03:45,940 --> 00:03:43,970 use like multiple sequence alignment or 83 00:03:49,119 --> 00:03:45,950 like a structural element in order to 84 00:03:51,789 --> 00:03:49,129 reduce the size of the library search 85 00:03:54,009 --> 00:03:51,799 pepper criteria is that this target 86 00:03:56,199 --> 00:03:54,019 should be maintained like the should 87 00:03:58,599 --> 00:03:56,209 have like the early amino acid in the 88 00:04:00,670 --> 00:03:58,609 most critical position for the function 89 00:04:02,110 --> 00:04:00,680 or for the structure this is quite 90 00:04:03,670 --> 00:04:02,120 obvious because for instance if we 91 00:04:07,150 --> 00:04:03,680 selected I don't know like an enzyme 92 00:04:08,800 --> 00:04:07,160 with the arginine in the one arginine or 93 00:04:11,199 --> 00:04:08,810 lysine in the active site if we 94 00:04:13,379 --> 00:04:11,209 substitute the arginine lysis is really 95 00:04:16,870 --> 00:04:13,389 probable that the protein will lose the 96 00:04:19,330 --> 00:04:16,880 the activity so we need to find also 97 00:04:21,819 --> 00:04:19,340 someone that maintain the amino acid in 98 00:04:24,120 --> 00:04:21,829 the critical position after that we need 99 00:04:26,379 --> 00:04:24,130 the target is well characterized and 100 00:04:28,540 --> 00:04:26,389 with the possibility to have like a 101 00:04:33,220 --> 00:04:28,550 selection method so to select the mutant 102 00:04:35,410 --> 00:04:33,230 so to perform very practically and 103 00:04:37,960 --> 00:04:35,420 the best target it was chosen was like 104 00:04:41,110 --> 00:04:37,970 the RNA binding domain of the ribosome 105 00:04:43,030 --> 00:04:41,120 betrothal at 11:00 this this domain is 106 00:04:46,900 --> 00:04:43,040 composed of seven three smaller domain 107 00:04:49,480 --> 00:04:46,910 76 amino acid so correspond to over the 108 00:04:53,170 --> 00:04:49,490 first criteria is like its function is 109 00:04:58,060 --> 00:04:53,180 to bind the specific sequence of RNA 110 00:04:59,740 --> 00:04:58,070 from the ribosomal RNA it's a 111 00:05:01,810 --> 00:04:59,750 conservative course it's a protein of 112 00:05:04,660 --> 00:05:01,820 the ribosome so is strictly conserving 113 00:05:07,270 --> 00:05:04,670 in most of species and in most of the 114 00:05:10,090 --> 00:05:07,280 species and in particularly the amino 115 00:05:13,020 --> 00:05:10,100 acid it arguing involved within the 116 00:05:16,030 --> 00:05:13,030 interaction with the DNA are mostly 117 00:05:17,710 --> 00:05:16,040 eliminated except for a recent lysine 118 00:05:20,020 --> 00:05:17,720 arginine but most of them are like a 119 00:05:24,700 --> 00:05:20,030 glycine Sarah so it's really good 120 00:05:27,730 --> 00:05:24,710 candidate for the but also if it's small 121 00:05:30,910 --> 00:05:27,740 protein still the library will be huge 122 00:05:33,520 --> 00:05:30,920 like we can talk like ten to the power 123 00:05:35,860 --> 00:05:33,530 of 30 variant if we won't substitute all 124 00:05:38,470 --> 00:05:35,870 the late amino acid with the early one 125 00:05:40,930 --> 00:05:38,480 so in order to reduce this one we have 126 00:05:43,170 --> 00:05:40,940 perform like different multiple sequence 127 00:05:46,450 --> 00:05:43,180 alignment or also structural analysis 128 00:05:48,850 --> 00:05:46,460 for instance we can we can reduce the 129 00:05:51,010 --> 00:05:48,860 size of library for instance is in IBD 130 00:05:53,410 --> 00:05:51,020 for in our target protein for this 131 00:05:55,560 --> 00:05:53,420 innocence pot could be like an arginine 132 00:05:58,630 --> 00:05:55,570 but we can find that in a really 133 00:06:02,050 --> 00:05:58,640 collagen product protein that could be 134 00:06:03,970 --> 00:06:02,060 like a Spartacus II that and these 135 00:06:05,950 --> 00:06:03,980 mutations to maintain the function so we 136 00:06:08,170 --> 00:06:05,960 can reduce the size of library in this 137 00:06:10,510 --> 00:06:08,180 way just with a multiple sequence 138 00:06:12,340 --> 00:06:10,520 alignment all for instance depend on the 139 00:06:13,660 --> 00:06:12,350 position of the late amino acid for 140 00:06:16,030 --> 00:06:13,670 instance if a late amino acid the 141 00:06:18,610 --> 00:06:16,040 present is like an alpha helix we can 142 00:06:20,950 --> 00:06:18,620 for instance avoid like the presence of 143 00:06:23,050 --> 00:06:20,960 a protein of prolene or other kind of 144 00:06:23,740 --> 00:06:23,060 immunity at the end of this analysis we 145 00:06:26,920 --> 00:06:23,750 are fine 146 00:06:29,140 --> 00:06:26,930 the we are reaching like the size of 10 147 00:06:32,260 --> 00:06:29,150 to the power of 10 that is still huge 148 00:06:34,780 --> 00:06:32,270 number but with the the mRNA display 149 00:06:38,290 --> 00:06:34,790 technique it's still possible to express 150 00:06:40,060 --> 00:06:38,300 this kind of library this technique is 151 00:06:41,620 --> 00:06:40,070 really powerful because respect to the 152 00:06:43,060 --> 00:06:41,630 other one for instance like the page 153 00:06:45,880 --> 00:06:43,070 display or the technique the technique 154 00:06:47,150 --> 00:06:45,890 that you can express a library maximum 155 00:06:49,040 --> 00:06:47,160 10 to the power of nine 156 00:06:53,480 --> 00:06:49,050 clone in these cases we can express 157 00:06:54,890 --> 00:06:53,490 express a 10 to the power 3 so 3 158 00:06:58,520 --> 00:06:54,900 magnitude more respect of the other 159 00:07:00,170 --> 00:06:58,530 technique this technique use it 160 00:07:02,420 --> 00:07:00,180 practically connect together the 161 00:07:05,210 --> 00:07:02,430 genotype to the phenotype so we have 162 00:07:06,080 --> 00:07:05,220 like the RNA the protein linked to its 163 00:07:07,910 --> 00:07:06,090 mRNA 164 00:07:11,060 --> 00:07:07,920 how it's possible thanks to the 165 00:07:12,590 --> 00:07:11,070 promising and these molecules so during 166 00:07:14,480 --> 00:07:12,600 the translation the ribosome when 167 00:07:17,060 --> 00:07:14,490 arrived to the three terminal interact 168 00:07:20,180 --> 00:07:17,070 with the promising they enter inside the 169 00:07:22,460 --> 00:07:20,190 ribosome a link the protein and in same 170 00:07:26,450 --> 00:07:22,470 time disassembly the ribosome so we have 171 00:07:28,820 --> 00:07:26,460 like the RNA linkage to the protein but 172 00:07:30,860 --> 00:07:28,830 before to perform the before to perform 173 00:07:33,950 --> 00:07:30,870 the mRNA display technique we have to 174 00:07:35,750 --> 00:07:33,960 optimize the selection method before the 175 00:07:40,400 --> 00:07:35,760 protein and we have optimized with the 176 00:07:41,930 --> 00:07:40,410 with the wall type so we use it like a 177 00:07:43,850 --> 00:07:41,940 cell free expression system so it's 178 00:07:46,510 --> 00:07:43,860 meaning that we didn't use the cell but 179 00:07:49,730 --> 00:07:46,520 just the extract of ribosome and all the 180 00:07:52,100 --> 00:07:49,740 translation I keep my fist timer for 181 00:07:54,680 --> 00:07:52,110 this patient so we will Express where 182 00:07:56,270 --> 00:07:54,690 the the protein were expressing was 183 00:07:59,540 --> 00:07:56,280 pricey and after that were incubated 184 00:08:02,810 --> 00:07:59,550 with the target RNA it was functional ID 185 00:08:05,920 --> 00:08:02,820 with a biotin molecules after that for 186 00:08:09,200 --> 00:08:05,930 the selection we can was like the 187 00:08:10,940 --> 00:08:09,210 complex protein and target so when 188 00:08:13,580 --> 00:08:10,950 incubated with the strategy bits so it's 189 00:08:15,650 --> 00:08:13,590 possible to select the the complex and 190 00:08:17,840 --> 00:08:15,660 here we can see like the reservoir like 191 00:08:19,610 --> 00:08:17,850 job well like in the first Lane we can 192 00:08:24,020 --> 00:08:19,620 see like the fluid row so all the 193 00:08:25,700 --> 00:08:24,030 protein that doesn't bind the tRNA and 194 00:08:29,090 --> 00:08:25,710 it's empty so it's meaning that all 195 00:08:32,180 --> 00:08:29,100 protein interact with the RNA with the 196 00:08:34,700 --> 00:08:32,190 target RNA in the washing step so it's a 197 00:08:36,529 --> 00:08:34,710 nothing so it's meaning that no we can 198 00:08:39,409 --> 00:08:36,539 bind there so all the protein binded an 199 00:08:41,810 --> 00:08:39,419 illusion you can see all our product it 200 00:08:44,360 --> 00:08:41,820 means that this protein this selection 201 00:08:48,650 --> 00:08:44,370 method is at least for the what type is 202 00:08:50,330 --> 00:08:48,660 tiwa be able so we have told you we have 203 00:08:54,310 --> 00:08:50,340 introduced with this selection method 204 00:08:57,200 --> 00:08:54,320 it's inside the Emily display technique 205 00:08:59,240 --> 00:08:57,210 here is like how to explain just let the 206 00:09:01,319 --> 00:08:59,250 floor show the the flowchart of the of 207 00:09:04,170 --> 00:09:01,329 this technique 208 00:09:07,490 --> 00:09:04,180 start with the DNA so we have like the 209 00:09:11,069 --> 00:09:07,500 DNA library after that we transcribe the 210 00:09:14,220 --> 00:09:11,079 DNA library RNA after that there will be 211 00:09:18,660 --> 00:09:14,230 the step where the RNA will be linked it 212 00:09:20,460 --> 00:09:18,670 to the promise in molecules and in the 213 00:09:23,009 --> 00:09:20,470 linker there will be like a fluorescent 214 00:09:24,600 --> 00:09:23,019 probe that will be useful for the next 215 00:09:26,759 --> 00:09:24,610 step just to follow like the different 216 00:09:29,970 --> 00:09:26,769 paper in the presence of not of the 217 00:09:32,309 --> 00:09:29,980 protein after that after that we have 218 00:09:34,439 --> 00:09:32,319 this construct like RNA plus promising 219 00:09:36,240 --> 00:09:34,449 there will be the expression so the 220 00:09:38,639 --> 00:09:36,250 junction between like the protein and 221 00:09:41,309 --> 00:09:38,649 the hair na and after that we have like 222 00:09:45,120 --> 00:09:41,319 so this library composite of like 10 to 223 00:09:49,350 --> 00:09:45,130 the power to the 10 to power 10 variant 224 00:09:52,199 --> 00:09:49,360 that will be selected toward the target 225 00:09:55,019 --> 00:09:52,209 RNA after that there will be like the 226 00:09:57,509 --> 00:09:55,029 aleutian and this type of reverse 227 00:09:59,699 --> 00:09:57,519 transcription because here we have DNA 228 00:10:02,999 --> 00:09:59,709 in this step we need the DNA for the 229 00:10:05,460 --> 00:10:03,009 sequencing or a new round of all to 230 00:10:06,840 --> 00:10:05,470 perform a new round of selection so 231 00:10:10,949 --> 00:10:06,850 there will be like just read less 232 00:10:12,360 --> 00:10:10,959 transcription so from DNA RNA DNA it's 233 00:10:14,249 --> 00:10:12,370 important also the negative control 234 00:10:16,170 --> 00:10:14,259 because I don't know it's someone worked 235 00:10:19,650 --> 00:10:16,180 with the RNA but the RNA is really 236 00:10:22,499 --> 00:10:19,660 sticky so we have to subtract the noise 237 00:10:24,120 --> 00:10:22,509 from the from the of the project so more 238 00:10:26,179 --> 00:10:24,130 or less for the negative control is the 239 00:10:28,439 --> 00:10:26,189 same step is the same process a 240 00:10:29,670 --> 00:10:28,449 parameter the only difference that to 241 00:10:32,910 --> 00:10:29,680 the approach the library will be 242 00:10:35,670 --> 00:10:32,920 incubated not with the the target RNA 243 00:10:37,439 --> 00:10:35,680 but just with the bits so in this way we 244 00:10:40,259 --> 00:10:37,449 can select it that the project that bind 245 00:10:44,100 --> 00:10:40,269 us specifically the the surface or the 246 00:10:48,360 --> 00:10:44,110 streptavidin or like the complex and 247 00:10:50,429 --> 00:10:48,370 after that is the same okay here we can 248 00:10:52,110 --> 00:10:50,439 see step by step because the mmm display 249 00:10:54,090 --> 00:10:52,120 respect to other technique is not 250 00:10:56,639 --> 00:10:54,100 something like a commercial a commercial 251 00:10:59,490 --> 00:10:56,649 key we need to perform and to optimize 252 00:11:02,939 --> 00:10:59,500 every single step here is the first step 253 00:11:05,340 --> 00:11:02,949 so the link the legation between the RNA 254 00:11:06,840 --> 00:11:05,350 and pure marking the promise is actually 255 00:11:09,540 --> 00:11:06,850 contain a fluorescence molecule so we 256 00:11:11,879 --> 00:11:09,550 can visualize in in fluorescence and we 257 00:11:14,130 --> 00:11:11,889 can see like the free pure attack so and 258 00:11:18,180 --> 00:11:14,140 in the the top of the part like the 259 00:11:20,040 --> 00:11:18,190 congregated MRNA to the to the to the 260 00:11:22,500 --> 00:11:20,050 porta marking on the other side we can 261 00:11:24,210 --> 00:11:22,510 see the visualization with like the 262 00:11:26,310 --> 00:11:24,220 common would be visualization with the 263 00:11:28,650 --> 00:11:26,320 generate and we can see that the the 264 00:11:30,990 --> 00:11:28,660 uilt it's more than eighty percent its 265 00:11:34,519 --> 00:11:31,000 meaning that still some RNA does bind 266 00:11:38,370 --> 00:11:34,529 the demise it but the process feel okay 267 00:11:40,829 --> 00:11:38,380 after that the the error name will be as 268 00:11:43,560 --> 00:11:40,839 price it will be translated and here we 269 00:11:46,500 --> 00:11:43,570 can see the difference between before 270 00:11:49,199 --> 00:11:46,510 and after so all the mRNA and after the 271 00:11:52,079 --> 00:11:49,209 translation so we can see a shift data 272 00:11:55,819 --> 00:11:52,089 meaning that there is like protein plus 273 00:11:59,699 --> 00:11:55,829 mrna so very we there is like expression 274 00:12:01,920 --> 00:11:59,709 and after that the selection so here is 275 00:12:03,900 --> 00:12:01,930 represented like the Pluto so all the 276 00:12:08,100 --> 00:12:03,910 library all the variant mutant that are 277 00:12:10,800 --> 00:12:08,110 not able to bind the target RNA so loose 278 00:12:13,860 --> 00:12:10,810 lost the function and it's the most 279 00:12:16,199 --> 00:12:13,870 abundant of course because no holding or 280 00:12:17,940 --> 00:12:16,209 like protein are not able to bind we 281 00:12:20,490 --> 00:12:17,950 worship that so just to remove like the 282 00:12:22,769 --> 00:12:20,500 wicked binder and the Lewisham so just 283 00:12:25,530 --> 00:12:22,779 this one its meaning that some protein 284 00:12:28,199 --> 00:12:25,540 maintain the function so composite of 285 00:12:29,610 --> 00:12:28,209 all early immune system maintain the 286 00:12:32,550 --> 00:12:29,620 function to interact with the target 287 00:12:34,560 --> 00:12:32,560 renege after that there will be the 288 00:12:36,540 --> 00:12:34,570 reverse transcription after the reverse 289 00:12:38,220 --> 00:12:36,550 at ratio this is like the visualization 290 00:12:40,110 --> 00:12:38,230 with the best prescription show after 291 00:12:42,780 --> 00:12:40,120 the aleutian will be retro transcribe 292 00:12:44,160 --> 00:12:42,790 and we are completed like the negative 293 00:12:48,000 --> 00:12:44,170 control the same drop from the negative 294 00:12:49,829 --> 00:12:48,010 control so without tRNA target and the 295 00:12:51,300 --> 00:12:49,839 library we can see at the beginning 296 00:12:53,490 --> 00:12:51,310 right round by round because was 297 00:12:56,670 --> 00:12:53,500 performing like 60-pound with times 298 00:12:59,130 --> 00:12:56,680 there was like a noise but after several 299 00:13:00,900 --> 00:12:59,140 time we start to decrease sensible the 300 00:13:03,360 --> 00:13:00,910 noise but still there so it's meaning 301 00:13:05,910 --> 00:13:03,370 that we there is like a subpopulation or 302 00:13:09,540 --> 00:13:05,920 protein that does not bind at the target 303 00:13:11,160 --> 00:13:09,550 RNA but only the bits but to understand 304 00:13:14,819 --> 00:13:11,170 which which was like this kind of 305 00:13:17,850 --> 00:13:14,829 sequence we have we have a sequencing 306 00:13:20,280 --> 00:13:17,860 the library at the negative control hey 307 00:13:21,630 --> 00:13:20,290 add the result of the from the neck the 308 00:13:25,980 --> 00:13:21,640 sequencing from the next-gen sequencing 309 00:13:28,199 --> 00:13:25,990 and ok of course the best binder was the 310 00:13:30,239 --> 00:13:28,209 water i don't know was how is possible 311 00:13:33,929 --> 00:13:30,249 there was like a contamination from the 312 00:13:36,720 --> 00:13:33,939 first round so the best bandit of course 313 00:13:39,059 --> 00:13:36,730 is the water but it at least its meaning 314 00:13:41,369 --> 00:13:39,069 that the method its raw booze I mean 315 00:13:44,069 --> 00:13:41,379 because the put post our maker is to 316 00:13:45,509 --> 00:13:44,079 select the best binder and the worst 317 00:13:49,280 --> 00:13:45,519 part of course is the best like that 318 00:13:51,389 --> 00:13:49,290 but here you see if we can find the 319 00:13:54,319 --> 00:13:51,399 contamination the noise you can see that 320 00:13:57,179 --> 00:13:54,329 around my round this this sequence is 321 00:13:59,609 --> 00:13:57,189 equal present about 19 the same 322 00:14:02,160 --> 00:13:59,619 abundance in the negative control and in 323 00:14:04,590 --> 00:14:02,170 the library so it mean that stick to the 324 00:14:06,809 --> 00:14:04,600 beat but we have found other three 325 00:14:09,569 --> 00:14:06,819 sequence especially the sequence number 326 00:14:12,540 --> 00:14:09,579 nine that is a really really good ratio 327 00:14:15,210 --> 00:14:12,550 between library so the samples with the 328 00:14:18,090 --> 00:14:15,220 RNA and the control negative control 329 00:14:19,919 --> 00:14:18,100 that mean that this this sequence this 330 00:14:22,319 --> 00:14:19,929 protein does not have the tendencies to 331 00:14:24,569 --> 00:14:22,329 bind the naked intruder to the beat so 332 00:14:26,819 --> 00:14:24,579 it's really probable that this protein 333 00:14:32,509 --> 00:14:26,829 is still buying all the mounting the 334 00:14:38,340 --> 00:14:36,269 here we have like Alana align the there 335 00:14:40,350 --> 00:14:38,350 was like the alignment of the different 336 00:14:42,509 --> 00:14:40,360 the selected protein respect to the work 337 00:14:46,530 --> 00:14:42,519 type and we can find that the Sam 338 00:14:48,809 --> 00:14:46,540 mutation welcome on in the different 339 00:14:51,259 --> 00:14:48,819 clue so for instance in some cases 340 00:14:54,359 --> 00:14:51,269 really interesting for instance like in 341 00:14:56,609 --> 00:14:54,369 there was like a substitution presented 342 00:15:00,269 --> 00:14:56,619 in the late 14th like the adenine 343 00:15:02,160 --> 00:15:00,279 welcome well substitute by aspartic acid 344 00:15:08,400 --> 00:15:02,170 so there was a completely a change of 345 00:15:10,199 --> 00:15:08,410 charge in an abandoned furthermore they 346 00:15:11,210 --> 00:15:10,209 were the during the reverse 347 00:15:14,280 --> 00:15:11,220 transcription 348 00:15:17,730 --> 00:15:14,290 there were also were inserted other 349 00:15:21,629 --> 00:15:17,740 mutation we can see that for instance in 350 00:15:24,600 --> 00:15:21,639 the the red arrow there should be like 351 00:15:26,460 --> 00:15:24,610 the well substituted like later early 352 00:15:27,989 --> 00:15:26,470 amino acid with other elimination this 353 00:15:30,210 --> 00:15:27,999 is spontaneous process during the 354 00:15:33,019 --> 00:15:30,220 reverse transition prescription but this 355 00:15:36,269 --> 00:15:33,029 mutation will well still maintain it and 356 00:15:38,669 --> 00:15:36,279 still maintain at least years like this 357 00:15:40,559 --> 00:15:38,679 is previous just homology modeling so we 358 00:15:42,269 --> 00:15:40,569 can see that at least it's the first 359 00:15:44,760 --> 00:15:42,279 analysis seems the 360 00:15:47,970 --> 00:15:44,770 the the the mutant maintain the function 361 00:15:49,560 --> 00:15:47,980 it seems that Monty also the the 362 00:15:51,360 --> 00:15:49,570 structure but for the moment is a 363 00:15:54,840 --> 00:15:51,370 previous data is just Amala G modeling 364 00:15:57,060 --> 00:15:54,850 so okay in conclusion the future work 365 00:16:00,150 --> 00:15:57,070 will be like of course the expression of 366 00:16:02,940 --> 00:16:00,160 the single protein characterization just 367 00:16:05,760 --> 00:16:02,950 to understand the binding constant with 368 00:16:08,850 --> 00:16:05,770 the target RNA and of course the the 369 00:16:11,160 --> 00:16:08,860 crystal structure on with the RNA so in 370 00:16:14,269 --> 00:16:11,170 conclusion we have identified a robust 371 00:16:17,610 --> 00:16:14,279 target for the reverse evolution project 372 00:16:20,430 --> 00:16:17,620 was customized the amenities the mRNA 373 00:16:22,610 --> 00:16:20,440 display technique for the best best 374 00:16:26,040 --> 00:16:22,620 mutant selection and it was identified 375 00:16:29,370 --> 00:16:26,050 three different protein able to bind the 376 00:16:33,120 --> 00:16:29,380 RNA also in presence of only early amino 377 00:16:35,670 --> 00:16:33,130 acid i'd like to thanks of my group from 378 00:16:38,640 --> 00:16:35,680 Chester University and particularly 379 00:16:40,550 --> 00:16:38,650 professor cosmic evolution from the SE 380 00:16:58,710 --> 00:16:40,560 in Japan for the collaboration and 381 00:17:01,500 --> 00:16:58,720 thanks to you questions very so you 382 00:17:03,180 --> 00:17:01,510 identified as a candidate this protein 383 00:17:05,309 --> 00:17:03,190 because it had a bunch of the early 384 00:17:09,059 --> 00:17:05,319 amino acids that important positions are 385 00:17:10,640 --> 00:17:09,069 there classes of protein that have much 386 00:17:13,919 --> 00:17:10,650 that have a larger tendency to have 387 00:17:16,380 --> 00:17:13,929 important sites Phoebe's early ones 388 00:17:18,390 --> 00:17:16,390 versus later ones no no they are protein 389 00:17:21,030 --> 00:17:18,400 that contain only early amino acid and 390 00:17:23,280 --> 00:17:21,040 we can cluster in some in some part but 391 00:17:25,380 --> 00:17:23,290 all the sequencer that we have selected 392 00:17:26,669 --> 00:17:25,390 contain only early memories know what I 393 00:17:29,700 --> 00:17:26,679 mean what I mean is when you look when 394 00:17:32,040 --> 00:17:29,710 you're looking at existent proteins I 395 00:17:33,380 --> 00:17:32,050 mean like like so you're saying there 396 00:17:35,880 --> 00:17:33,390 are ones that have there are existing 397 00:17:39,450 --> 00:17:35,890 proteins that you can grab say that only 398 00:17:41,669 --> 00:17:39,460 have in the pretty well it was plausible 399 00:17:44,100 --> 00:17:41,679 that was some protein like that right 400 00:17:46,320 --> 00:17:44,110 but no evidence I mean it was just 401 00:17:48,900 --> 00:17:46,330 basing on the like a simple Inga like on 402 00:17:51,030 --> 00:17:48,910 the mater I tend on the prebiotic food 403 00:17:52,960 --> 00:17:51,040 but no protein was fine that contains 404 00:17:54,669 --> 00:17:52,970 only eliminating 405 00:17:56,980 --> 00:17:54,679 there are if there are modern protein 406 00:17:58,840 --> 00:17:56,990 classes that are more biased towards 407 00:18:00,850 --> 00:17:58,850 early amino acids than later today I 408 00:18:03,250 --> 00:18:00,860 mean if there are functions in the cell 409 00:18:20,600 --> 00:18:03,260 that seems that way what do I mean no 410 00:18:25,159 --> 00:18:23,000 thank you for your presentation I 411 00:18:27,680 --> 00:18:25,169 realize that you already have a lot of 412 00:18:29,720 --> 00:18:27,690 possible combinations in your essay when 413 00:18:32,029 --> 00:18:29,730 you start but I was wondering if you 414 00:18:34,190 --> 00:18:32,039 ever considered adding prebiotic 415 00:18:37,009 --> 00:18:34,200 irrelevant amino acids that we do not 416 00:18:40,940 --> 00:18:37,019 find in proteins nowadays for example or 417 00:18:42,529 --> 00:18:40,950 anything could sort solve the problem 418 00:18:45,200 --> 00:18:42,539 that right now you're not able to use 419 00:18:47,659 --> 00:18:45,210 any positively charged amino acids yeah 420 00:18:49,730 --> 00:18:47,669 in this is the first step so we have 421 00:18:51,889 --> 00:18:49,740 selected only the early amino acid 422 00:18:53,930 --> 00:18:51,899 present in the genetic code but of 423 00:18:56,269 --> 00:18:53,940 course the most Theory provide like 424 00:18:58,399 --> 00:18:56,279 evidence that was like composite the 425 00:19:01,279 --> 00:18:58,409 early amino acid of like conventional 426 00:19:03,379 --> 00:19:01,289 it's like the modern one so like the 427 00:19:05,750 --> 00:19:03,389 present is but also non-conventional one 428 00:19:07,549 --> 00:19:05,760 forest and one of the cases like the old 429 00:19:10,159 --> 00:19:07,559 meeting in fact if we notice the the 430 00:19:12,049 --> 00:19:10,169 table was not positive charged amino 431 00:19:14,269 --> 00:19:12,059 acids quite weird so it's possible that 432 00:19:15,889 --> 00:19:14,279 with the ornithine we replace it after 433 00:19:18,019 --> 00:19:15,899 that one so but there will be the next 434 00:19:20,090 --> 00:19:18,029 step of the project so first up like 435 00:19:21,740 --> 00:19:20,100 this one and after that we see two 436 00:19:23,870 --> 00:19:21,750 looking for the non-conventional but 437 00:19:25,570 --> 00:19:23,880 this is a quite difficult because you 438 00:19:27,620 --> 00:19:25,580 need to integrate in the like 439 00:19:29,470 --> 00:19:27,630 translation system the possibility to 440 00:19:32,690 --> 00:19:29,480 insert non-conventional amino acid is 441 00:19:35,169 --> 00:19:32,700 it's not so easy to modify the tRNA 442 00:19:37,750 --> 00:19:35,179 synthetase G and blah blah blah 443 00:19:40,940 --> 00:19:37,760 thank you very much for your talk I 444 00:19:43,549 --> 00:19:40,950 assume that the positive charges in the 445 00:19:47,090 --> 00:19:43,559 proteins are interacting with a negative 446 00:19:50,750 --> 00:19:47,100 charge of the phosphate in the renamed 447 00:19:52,639 --> 00:19:50,760 and but in your new proteins several of 448 00:19:56,000 --> 00:19:52,649 those positive charges are genes and 449 00:19:57,100 --> 00:19:56,010 license are now negative like in 450 00:19:59,990 --> 00:19:57,110 something 451 00:20:03,379 --> 00:20:00,000 aspartic acid do you have any idea or 452 00:20:05,649 --> 00:20:03,389 any model of how now these negative 453 00:20:10,430 --> 00:20:05,659 amino acids are interacting with with 454 00:20:12,409 --> 00:20:10,440 okay thank you in the most the only 455 00:20:14,629 --> 00:20:12,419 parts that interact with the DNA is 456 00:20:24,289 --> 00:20:14,639 particularly these alpha Alex and this 457 00:20:26,419 --> 00:20:24,299 loop and mostly and and this alpha helix 458 00:20:29,210 --> 00:20:26,429 and this loop is covered in this part so 459 00:20:32,120 --> 00:20:29,220 this remain is remaining it practically 460 00:20:34,310 --> 00:20:32,130 the same and probably these when it's 461 00:20:36,140 --> 00:20:34,320 like for this this charge 462 00:20:38,420 --> 00:20:36,150 on the other part of the Alfa Alex so 463 00:20:42,080 --> 00:20:38,430 it's not interact directly with the tRNA 464 00:20:46,010 --> 00:20:42,090 so probably is not important for that 465 00:20:47,660 --> 00:20:46,020 binding and also it's also possible that 466 00:20:50,690 --> 00:20:47,670 I don't know maybe some positive charge 467 00:20:54,440 --> 00:20:50,700 like the KT or cation can can function 468 00:20:56,870 --> 00:20:54,450 like like a bridge so it's also possible 469 00:20:58,400 --> 00:20:56,880 but I know this is only a monetary model 470 00:21:01,100 --> 00:20:58,410 in fact next step would be to have the 471 00:21:03,890 --> 00:21:01,110 crystal with the target RNA it wanted us 472 00:21:08,630 --> 00:21:03,900 to know how it's like fit so future warp 473 00:21:09,480 --> 00:21:08,640 I hope that will be soon okay let's take