Your  arguments. Welcome  back  to  the  last  section  of this  domestic  meeting  and the  section  of  the  city. I'd  like  to  use  myself  from the  University  of  the  section and  and  the  sections, the  first  one  my  myself  and  save  showing how  exclude  DNA  added  to resogions  and  alter  the  DN  averse. And  in  the  second  be parado  her  system  she  developed  using Boyd  to  actually  learn  more  about Ss  signaling  And  in  the  Herst, in  the  studies on  X  and  loss, which  we  know  is  really  chemosis. Not  very,  I  really  look forward  to  learning  more  about  this, talking  about  crops, we  did  very  important discoveries  in  the  past. Microns, about  a  new  techniques developed  to  understand more  about  this  An  and  the last  developed  Phys  Gamine  the  Frisby. And  he's  going  to  be  talking  about how  the  developed  system  using  Pbgd  make individual  gene  engineering  and last  as  the  layer. And  he's  going  to  be  talking  about  his  work, how  he  optimized  the  musical  game  system. He  made  very  efficient  pins  and  jeans. So  this  sport  and  let  me start  mine  and  work  was  dot  by  attendant, she  was  held  by  flashy. And  this  system  is  action  by  most  obvious, beautiful  work  that  sissy  guided  signal static  and  the  first  one  which  was discovered  and  the  tax  pates was  the  physical  gas  line. And  we  know  that  it may  blows  in  the  Dna  base, but  it's  not  always  the  case. It's  been  shown  previously  in  animals. And  as  that  cans  make similarly  over  times,  rapidly, recently  and  recently  implants  to  show that  can  also  make  starts  and  the  Pac, these  cats  and  and  Nat  have  to  be dependent  on  the  Gic  context of  the  site  if  you'd  like  to,  multiple. So  as  a  take  usually  in  Comcast, there  is  another  recently  discovered spenser  clip which  make By  second  vs  the  DN  base. What  happens  after  rev the  levels  in  the  DNA  rigs  itself, it  alerts  itself  on  DNA  shines  and one  of  the  most  active  ones in  visit  the  Ces  is the  land  pathway  which  is  tries  to  slip together  the  broken  VA.  And  if it  doesn't  work  and  the  ends  can be  exposed  to  the  places  and then  they  can  ape  to  micrologycausehlesssdes. The  pathway  is  verticals, micromole  or  automation  pathway, beginning  and  get  further  reset and  also  be  involved  in  HR. But  will  be  news  normal, engage  the  autogenic  pathway  and the  position  the  Desford  to  use. We  employ  the  reporter. A  simple  and  very useful  physically  target  and  indication. A  loss  of  popping,  addition  in acid,  resulting  globalizing  colors. We  can  the  cells  comps and  we  can  recover  all  cells  very easily  with  some  NA and  the  charge  exploration, introducing  the  Excel  and give  them  on  globalizing  plates  and  nobis, pates  and  we  the  frequency of  editing  as  percentage  of  Biz  cells. We  also  poles  what is  happening  in  these  sides. Imagine  the  Cd  makes the  staggered  cuts  and  get  farther  sequence, but  you  appreciate  probably  it's  quite similar  to  the  cuts you  can  make  with  station  size. What  you  do  with  one  cooling because  the  DNA  you  provide the  DNA  commentary  ends lug  together  you  figure  what  you  want. You  can  use  the  Cd  system to  gates  very  efficiently  just  by using  these  overhangs  that  its  to the  S  with  the  Cdm and  then  add  the  short  pads. So  these  are  poles  in the  DNA  containing  pole  frames. And  they  have  overhangs  to the  Cdc  side  that hopefully  we  can  move this  sequence  efficiently. Into  the  Cdpap  was  just  CP  one  RB  on, and  as  T  is  not  very  efficient, usually  we  can  add  over  5%  itself. In  the  experiment  we  just added  it  results  in  editing, but  when  we  stood the  Palm  is  actually  the  frequency  of editing  went  up  from almost  4%  to  47%  first  on  this  plate. And  when  we  did  the  okaying, efficient  looking  system  and  before  we  want to  champagne  as  my  student  look  statement, what  is  happening,  uh,  in  this  code  size? And  it  was  applies  only  a  few  molecules we  integrated  into  the  Cots. It  was  unexpected. And  then,  and  because  of this  one  was  limited  by  gene  and the  experiments  and  focus on  the  determine  what happens  if  we  just  change the  terminal,  the  shoulders. And  with  the  Npcs, we  play  with  the  fibers  with Atb  three  movable  and plans  the  Ssds  of  them, the  short  moles  able  to boost  the  fitness  of anything  very,  very  efficient. Then  we  obviously  sequence all  of  ponies  from  independent  discernment. You  can  see  here  in  the  To. These  are  experiments  with  R  and  B, only  what  you  recover, the  lows  of  deletions  and  insertions, and  this  virtue  is  in  ponies. We  also  added  the  shortened  dealing  workers. 5.3  Bible  Warning,  it's  quite  similar. We  use  plebs,  with  blonds, we  do  more  insertions and  indeed  when  we  analyze the  sequences  in  Del, we  found  that  most  insertion, what  we  found  here  derived from  the  voles  we  want  to, what  we  concluded  from  this is  that  the  oleic, those  movements  with  5.3  Bibles  are  not very  efficient  in  boosting integrated  indication. The  ones  with  blond  intergrate usually  has  Fps  then  size. So  696  double  the  size of  the  double  individual. And  I  did, my  overhangs  usually  adds  this  leak. This  you  can  see  here  a by  increasing  the  size each  port  from  five, which  did  not  really  boost  the  city, but  we  just  doubled  it. It  went  up  from  basically  over 5%  to  30%  lingual  cluster which  is  used  guides, the  toes  were  not  very efficient  and  the  foot  some  efficient section  as  the  Tled  ones and  then  we  pop  together. So  these  are  the  double  standard ones  and  these  are the  class  size  sided  ones  that's  efficient, located  might  be  big  secondary  structure. When  you  use  the  four  programs, you  always  find  secondary  structures. And  because  of  this  late  use legal  which  doesn't. Second  for  example,  policy  and  indeed  we used  more  structured  do it  didn't  boost  the  frequency,  medicate. And  then  what  we wanted  to  test  is  what  happens. Candied,  if  we  made  the  Legos  resistant  to nucleases  and  infield  use  these  bonds, madest  nucleases, this  is  what  we  added  to  the  Dei, and  what  we  found  is  that  when  we added  these  post  bonds, they  completely  acknowledged the  Dicot  medicate. In  contrast,  when  we added  the  modifications  to  the  middle I  actually  starting  is frequency  affected  from  this. What  we  concluded  that  the  terminals  are important  and  it's likely  that  terminal  medical, so  currently  there  is  no  known  mechanism for  this  but  we  have  uses. What  we  think  is that  when  we  have  a  send  the  break, then  the  rear  machinery identifies  it  and  get  reputed. But  if  you  have  access  of  DN  in  the  cell, then  the  rear  machinery actually  gets  sequenced  away from  the  physical  outside  the  end, the  DN  can  act  as  a  Po, and  we  do  hypothesis. We  employed  different  dynamics. We  generated  mutants  in  the  major, those  in  the  DNA  pathways and  obviously  the  Galena, because  it  been  established  that  the combined  was  in  the  DNA  temperatures. And  we  also  generated mutant  pathway  in  the  detention  pathway, which  assesses  DNA  in  the  Nclogy  pathway, and  then  tested  what  happens  in  the  mutants. And  what  we  found  is  that  this  is  the, all  this  is  the  boost, we  see  these  oligos  regions  and  in  the  mid, even  below  adding  the  oligos  behaviors  being record  what  it  showed  is that  is  likely  to  be  involved  in  this  part. And  then  we  added  nuclear types  types  to  this  giant. Together  with  this  vision, it  boosted  the  efficacy  of everything  suggesting  that  additional  factors might  also  be  involved. And  indeed  we  tested  the  medians. Boosting  effect  must  be  used  compared to  the  but  still  adding Obs  increase  the  frequency  of ethics  that  these  also  involved  in. No  Inh. Just  like  to  summarize  that, Nosings,  a  deploy  by sequencing  the  proteins  and imbalance  between  the  Ninos. This  is  funders  and  thank  you  much  for Postdates  of  DNA  process. Microcod  chance  to  how  big? When  we  haven't  nece  we haven't  tested  long  walks  because  the only  was  what  we  need  to  get applies,  the  long  defective. Ode. Thank  you  much.  Have  you  actually tested  the  effects  of the  concentrations  of  the. Yes.  So  probably  expected, so  it  has  nine  posters, so  it  was  like  maybe  ten  times. I  at  least  that. So  it  has  historical  conditions  like 0.5  to  two  medical  and there's  a  kind  of  concentration  response. Thank  you. We  tried  moles,  mismatches, but  these  are  actually  nobles. So  the,  the  Dobson  division Gd  have  nothing  to do  with  any  conditions.  That's  fair. I  mean,  mismatches  in  the  doubles  land, all  that  would  attract  additional  repair. Okay.  I  see  your  point. We  tried  Dobson  ones  with different  ty  structures  like loops  and  other  things  and  then  support. And  in  terms  of  efficiency, I  don't  know  my  heart  very similar  to  the  normal  defined  ones. I  think  it's  very  nice  to  see  that  maybe after  all  the  starting  us  and Picor  do  check  if  you  get  more, more  specific  integration  with the  small  pieces  rather  than  normal set  we  are  doing the  experimenter  sanos  the  front. The  additional  back. Yeah. It  seems  like  the  what the  oil  is,  it  doesn't  really  matter. Did  you  just  use  like  the  shear stands  for  DNNA  and  that  work? Yeah.  You  see  that? The  size  desk,  So  Okay. It  works  from  540, 5%  of  DNA  upwards. And  we  haven't  tested  like briefly  on  the  door  along  the  ones, so  Yeah,  But  in  theory,  yeah. Instead  of  wording  all  it  goes, you  use  the  jets. Yeah,  I  did. Thank  you  much. Can  you  hear  me? It's  great. Great. And  the  next  by? Yeah. Hello  everyone,  and  scoop. And  also  the  opportunity  to present  some  our  work  today. During  my  talk,  I'm  going  to present  how  we  review  the  proxies of  the  1.2  using  a  tool  based proximity  labeling  as  a  new  method to  map  protein  interaction  networks  in  the, the  role  of  the  vesicle  is using  protein  and  plastids. But  one  is  an  ongoing  question and  research  years, many  hypos  hypothesis  about expansion  being  involved  in titled  Bioassay  and  Coping  With  Man  Stress. So  it  was  already  found  in  apitosis, that  a  division  of  the  one  tos  of  Tyler. As  you  compare  for  example, the  type  with  a  knocked  down  urgent  and  oral out  of  the  one  which  has empty  lots  and  can  only  survive  from, but  one  is  not  only involved  in  Tyco  biogenesis. The  oligo  manifestation  of  the  one  in manoch  structures  is  catalyzed by  the  Hsp  70  chaperone  system. Together  with  the  chaperone  Ch  two  and  Cch. One  further  one  can  create  membrane  keywords to  transfer  lipids  from  the  outer below  to  the  Tyll  membrane. When  there  are  stressed. For  example,  when  there  is highlight  or  oxidated  stress, this  leads  to  an  imbalance  in the  protein  homeostasis.  What  results? For  example,  protein oxidation  or  toxic  aggregates. In  Moog  there  is  AP  one  parallel  named  two expressed  under  stress  condition and  Ebit  one  with  two  helium. That  involved  in  sense  and  coping  with membrane  stress  together  with molecular  chaperones  and  proteases. In  addition,  it  seems  that the  lives  are  involved  in  Posi. Now  our  question  was  which  protein works  together  with  the  Ops in  the  different  function? And  to  address  this  question, one  to  establish  proximity  labeling. In  general,  proximity  labeling is  based  on  a  fusion  of the  late  protein  here  in yellow  to  the  equally  in  red, which  activates  biotin  by adulation  while  at  P  is  consumed. In  the  next  step,  the  reactive  biotype, the  yellow  dots  normally  transferred  to a  specific  lysine  residue  of  the  aquinas. A  couple  of  years  ago,  by introducing  a  single  point  mutation, this  specificity  was  lost. The  reactive  biotin,  Alp  is  lease, and  can  then  attach  to  exposed  primary  lines of  proximal  proteins  in the  labeling  radius  of  ten  nanometer. Proximal  blue  and  creek  tal  proteins  equate, get  labeled  after cells  and  protein  extractions, the  inlaid  proteins  are enriched  straps  and  then  analyzed  them. Stratum  try. The  problem  with  this  original method  was  that  it  has a  labeling  time  is  up  to  18 H.  To  overcome  this  problem, several  more  mutations  were  introduced, resulting  in  Tv  that  achieves the  same  labeling  efficiency in  10  min  and  had a  slightly  bigger  labeling  radius  is  35  each. During  our  experiment,  we took  different  examples. On  the  one  hand  we  had the  imperfection,  these  are  holes. Before  incubation  of  the  Bs. We  also  took  a  blow  through  example for  Utd  proteins  and  after illusion  of  the  biotinylated as  the  lates  analysed  by MS.  As  you  already  heard  to  talk  of  Luke. You're  already  familiar  maybe  with the  system  as  it's  already also  a  parallel  established  in  parallel. At  the  beginning  of  our  project, we  used  the  modular  cloning  system to  fuse  our  proteins  with  the  two. As  we  were  interested  in  the  Ip  system 1.2  our  bits  with  the  Ctu  fusion, The  analysis  for  fusion  protein  accumulation using  antibody  against A  showed  a  strong  expression  for the  one  fusion  protein  impart. As  already  mentioned, important  to  choose  the  controls. On  the  one  hand  we  included  a  non transformed  Ubm  four  type  to  have a  control  to  separate  between  proteins bitulatedtuorfdogenictulated  protein and  contaminants  Ubi  four. We  observe  no  fusion  protein. As  a  second  control  we  chose an  Mary's  an  unrelated  protein. We  targeted  logs  and also  procedure  to  fusion. In  all  the  experiment  we  chose to  work  with  two  transformers by  then  expresses  the  to  statin  higher  level. Then  next  step  we  had to  find  the  optimal  concentration of  externally  added  tine and  sic  optimal  time. Therefore,  we  tested  different  conditions for  water  and  also  control. And  everything  was  analyzed by  17  coupled  days. On  the  one  hand,  we  tested without  applying  any  tine, but  we  also  tested  by  treating the  cells  with  Tine  over  a  time  period  of 24  H.  We  observed  without  adding  some  Ote, strong  Atul  cell  Byu  of one  and  also  additional  biotulatedtinesupon. Adding  tine,  the  signals increased  up  to  6  H,  but  not  further. When  the  cells  were  incubated  up  to  24  H, we  also  tested  different  concentration and  we  observed  enhanced botulationhen  treated  with  1  million  Mod and  became  more  or  less  to  the  same  size as  in  this  study. And  we  also  tested  the  Lim  four  words where  we  observed  no  increase  in timulation  when  we  incubated  the  cells with  up  to  18  H  compared  to  the  nonres. Now  for  our  Map  one, we  incubated  cells  with  1  million  molar  for 4  H  and  the  results  are  going  to show  you  the  one  Hetp Ft  and  on  the  other  hand, Oleds  focused on  the  one  hand  on  continuous  did, but  we  also  treated  as  cells  with hydrogen  peroxide  for  4  H in  parallel  to  reduce  stress. As  you  remember  that  the  Tress  and Cox  stress  in  the  input  fracture, we  observe  already  the  strong  cumulation again  after  the  one  too. And  it's  a  bit  hard  to  see, but  you  see  also  some  additional  bitlines. Both  conditions  in  the  strains, biotinylated  proteins are  then  strongly  enriched. In  the  dates,  under  both  conditions, in  all  of  the  five  strains. While  you  see  a  reduced pattern  in  the  Um  four, we  only  have  endogenous  bitumulated  proteins or  contaminants  based  on  the  immunoblot, We  observe  no  huge  differences  in the  bioaccumulation  pattern  or the  number  of  biotinylated  proteins when  we  compare  stress with  the  evolution  of  data  Shows  you  now. Comparison  of  the  protein  balance  after the  one  versus  the  cherry  line. After  the  subtraction  of the  contaminants  or  undergoing  proteins  from the  four  we  see  in  two  protein  and  the  one and  already  no  interaction  pattern  and green  like  H  seven  and  Cv  two. And  under stress  conditions  the  non  interaction 1-2  verified  as  well. We  found  several  proteins with  fraction  based  on our  study  we  named  the  proximity labeling  proteins. We  found  some  of  them  are already  at  hand  conditions, but  most  of  them  are  found under  the  stress  treatment. We  also  found  some  proteins  involved  in the  bioenergy  complexes  as  a  search  group. We  found  some  proteas that  are  probably  involved  in the  regulation  of  photosynthetic  electron taking  oocyte  and  two  together. There  are  proteins  found  in both  proxies  only  under  continuous  lab. For  example,  we  have  Chp  one  in both  Proxima  Pl  nine  only  in the  one  Pxi  or P  one  and  P  one  only  in  the  two  proxy. There  are  proteins  only found  under  stress  conditions. For  example,  VP  3.4  and  both  proxies. There  are  several  more  Ps only  under  stress  and  little  one  Proxim. We  also  identified  clip three  in  proximity  to  one. Under  stress  proteins  form in  both  proxies  and  above conditions  for  1.2  or  five  in  both  proxies. Now  as  a  group  of  concept, we  went  for  Sip  proximity labeling  to  confirm  some  of the  proteins  also  in proximity  to  B  one  hand  we chose  P  two  as  L  proteins with  an  structure  as  a  second  protein. Chose  Ptl  one  as  volutes involved  in  regulation of  proxy  electron  flow. Coming  to  a  plea  twos  novio, We  observed  already  in the  unilat  analysis  the  presence of  P  one  in  the  late  fraction  of  VP  two. But  you  see  that  the  signal  is  reduced  in the  liar  control  and  was  absent  in  the  four. This  was  also  verified  by  Ron  S  data, so  there  is  one  and  proximity  to  VP  two. In  addition,  we  found  two  other  proteins, plea  ten  and  P  three. They  were  also  identified  before. In  the  P  for  PT  one, we  only  found  one  in the  proximity  P  one  and  P  two. Mainly  or  probably  because the  study  was  performed  at an  condition  where  we  express, we  also  found  Pl  proteins and  also  some  proteins  involved  in the  bios  of  Y  protein  complexes and  these  were  also  found before  in  our  study. As  a  main  proof,  we  found  several  Tes, or  already  known  proteins that  are  involved  in the  regulation  photosynthetic  electron  flow or  specifically  in  the cyclic  electron  flow  complex. Take  out  our  results  together  successfully established  proximity  labeling  in the  class  using  the  Td. We  also  use  modal  system to  generate  our  constructs. To  use  our  Td, we  worked  in  early  experiments with  several  controls  that  we can  differentiate  between endogeneity  proteins  and  pots. But  also  we  had  to consider  positives  and  what's  negative. In  our  study,  yeah, what  we  now  did  was  to  try to  crop  interaction  network. We  found  proteins  that  are  involved in  the  biogenesis  of Nacl  113  or  Chf  one. We  also  found  the  shale  clip  history in  proximity  on  an  stress, we  found  many  annual  proteins  we call  the  Pl  media  stress. This  gives  us  an  idea  that  they  might  be involved  in  coping  or sensing  with  membrane  stress  as  well. Based  on  some  overlaps  between the  viboxium  and  the  Ptl  one  Bxi. There  might  be  also  that there  is  connection  between the  Vip  system  and  cyclic flow  if  you  want  to  try  proximity, labeling  in  your  lactates  are  also available  at  the  Clams  Center. At  the  end  of  my  talk,  I want  to  say  to  people  involved  in the  projects  or  supervisor  a  huge  steps, she  did  the  main  work  in  establishing  method. Melissa  just  and  were involved  in  performing  the  rest  of pop  experiment  and  static  under the  MS  measurement  as  well  as  the  funding. And  your  further  attention, I'm  wondering  whether  you  try, did  you  go  achieve? Why  back  compared  to  a  mutant  back? G  one  mutant? Or  mutant? Yeah.  So  at  the  moment  we're  just working  with  the  UBM  four  It  type  strain, but  we're  also  thinking to  change  the  background  so that  we  also  tried  to  do mutant  background  but  it's  not  yet  done. Do  you  know  what  is the  proportion  of  the  legible Id  one  versus  the  Gm  one? Because  I  guess  the  two. I  don't  know  that. Did  you  take  a  look  at  the  fusion  protein  and the  shares  the  core  somewhere? Yeah.  Actually,  what's  missing in  our  work  yet,  it's  the  localization. Also  for  our  proteins we  count  and  also  for  example, to  do  core  localization. But  when  we  go  back to  the  slide  we screen  for  positive  transformers, you  see  by  sharing we  often  observed  this  kind  of  tone. We  were  thinking  that  maybe  it  could  be some  precursor  proteins  or  that  it's not  fully  located  in  the  lost, maybe  it's  partially  also located  in  the  string. Therefore,  we  are  trying  to  get a  new  control  so  that  we go  away  from  arterias  control and  try  to  find  better  controls. Can  you  identify  the  Cies  which  video? The  ten  in  the  aspect. Can  I  tell  you  something about  decide  interactions between  and  those  in  general, the  Biotin  binds  crew  exposed  a  minus,  minus. And  it's  not  showing, or  the  whole  study  does  not show  you  a  clear  interaction. We're  just  talking  about proteins  in  a  specific  radius. The  method  does  not  show  you a  clear  interaction,  for  example, between  one  and  the Pls  that  they  directly  interact. It  just  shows  you. Ok,  at  this  specific  moment, we  find  these  proteins  in proximity  to  two  our  weights. Yeah,  I  was  wondering Baker  to  copies  which  are  membrane  copies. Membrane. And  then  look  at  because  Yeah, so  maybe  you  just  answer this  question  you  ordered  because that  many  copies  action you  actually  found  which  are  in the  membrane  ID  involved  in  Cyc. I  guess  when  we  started  the  project it  was  mainly  because  we  are working  a  lot  with  S  and  for the  reciprocal  experiment  or  proteins  that found  some  proteins  involved  proteins, Cedars,  other  sated  resalable proteins  in  the  stroma. But  we  also  have  some  data  in  our  publication where  we  perform the  tool  bid  on  extracted  membranes. So  that  is  also  a  second  approach that  we  have  in  the  publication. Another  question  from  the  label, that  contents  from  data, I  would  say  on  one  side, on  the  one  side  I  would  say  yes, on  the  other  side. I  would  say  it  depends  a  little  bit on  how  is  the  structure  of  your  protein and  how  many  lions are  there  where  labeling  can  happen. This  requirement  at  share is  also  known  to  Gilor  to  spit. Okay. Yeah. Let's the  next, there  is  hello. This  time  slot  at Louisiana  Saving  Therapy  is they  talk  to  me  about how  plan  sales  collectively migrate  during  pneumonia  cases. So  just  the  overview  of collective  migrations, movement  and  groups  that affect  individuals in  the  group,  their  behavior. It  can  be  used  to  optimize nutrient  conditions  as  it does  in  my  research. And  this  is  well  studied  in other  species  like  animals  and  bacteria, but  never  has  been reported  in  clammy  until  now. Also  well  studied  in clammy  is  the  photo  tactic  mechanism. You  see  this  example  is Pts  on  the  top  left  here, RCC  124  is  giving us  a  negative  photop  tactic  response. We  know  how  this  happens. We  know  that  there  are  channelled involved  in  detect  light  and  we  know the  pathway  that  follows that  to  induce  this  photo  tactic  response. But  for  example,  when  I  see a  positive  Pmo  tactic  response and  that  the  *****  population of  plans  bottom, entire  portion  of  the  chemical  breaking is  part  of  the  chemotactic  response. Nobody  knows  how  this  happens. There's  not  much  literature on  chemotactic  mechanism. What  receptors  are  involved, what  pathways  happen  after the  chemicals  are  detected. And  just  gotten  up. So  that  led  us  to  really  want  to investigate  this  chemotactic  mechanism. And  as  we've  seen  in  this  conference, there  are  a  lot  of  studies  on  phototaxis, how  it  happens,  and  there's a  lot  of  things  we  can  do  around  it, but  not  so  much  on  chemotaxis. So  me  and  Alex  are  the  only  ones talking  about  chemotaxis in  this  entire  conference. Right? Not  much  literature  exists. So  we  just  want  to  elucidate the  mechanism  that's  is  working  on. And  when  I  speak  about  chemotaxis, I'm  specifically  talking  about ammonium  because  there  are other  chemoattractants  that  are  study. But  we're  going  to  focus  on  ammonium  for our  research  just  to give  you  an  idea  of the  experiment  for  design. Because  this  is  a  modified  pet  dish  assay that  not  many  people use  in  literature  before. When  Chemotaxis  study  is  studied in  test  tubes  or  capillary  tubes, it's  not  really  able  to  be visualized  like  this  in  a  spatial  gradient. Let's  just  put  it  in  the  fetus, in  trog  latina. Then  we  create  auto  blocks  to create  our  chemical  gradient  from  the, we  go  from  no  nitrogen  to  excess  nitrogen. And  then  we  expose  it  to control  homogenous  light. Usually  white  light  where  we  can  change light  The  strains  that we're  going  to  be  talking  about  today. When  I  say  will  type, I'm  talking  about  CC  124 and  not  so  much  CC  125. And  just  to  point  out  that  it's  lacking that  Ag  one  gene  and  then  I  also  use  Co  53, you  know  that's  a  clip  parent  strain has  a  normal  Ag  one  gene. And  then  I  also  have  some  transgenic  strains, number  six  and  number  13 which  have  a  recombinant  age  gene. Just  to  say  like  we've  seen  in early  example  of  phototaxis, lacking  that  Ag,  one  gene  produces that  strong  negative  phototaxic  response that  we  saw  earlier. I  also  want  to  let  you  know  that  we  do these  experiments  over  24 h.  So  like  the  photo  tactics, these  experiments  are  done over  minutes,  maybe  5  min, 10  min,  but  I'm  looking  at  the  entire  24  H. The  kind  data  that  we  can  start  to  visualize from  that  assay  is  time. That's  video. So  you  see  here,  just  stop. You  see  here  a  positive  chemotactic  response as  it's  moving  from  top  to  bottom. And  we  also  did  a little  modeling  on  our  gradient. As  a  legend,  her  experiment  with Bet  just  to  quickly  show  this, so  we  know  our  division  coefficient and  we  know  that  our  gradient  is stable  over  that  full  24  h.  Since  we  are in  the  first  looking  at the  type  of  experiments  for  this  long, we  want  to  make  sure  that  the chemical  gradient  that  we're actually  creating  stable  over  time. And  also  a  way that  we  can  visualize  our  data. I  create  these  photost, look  at  them  a  036  to  over  24 h.  So  this  is the  same  feature  just  five  times. And  when  I  say  0  h, I  mean  the  moment  that  the  glocks  are placed  into  the  homogenous population  planning. The  first  result  I  want  to  show  you  is  that using  light  enhances  ammonium  chemotaxis. When  I  say  that  I do  these  experiments  in  light, usually  I've  done  them in  many  different  colors  of  light. And  I've  even  done  them  in  the  dark  in CC  124  and  just  so  this  photostat. So  over  time  you  can  see this  positive  chemotactic  response. Same  same  exact  experiment done  with  the  dark. Positive  chemotactic  response,  but  not  so, it's  not  so  aggregated. So  just  to  small suggest  that  light  is  enhanced  chemotaxis. And  then  we  also  develop a  way  to  quantify  the  intensity  over these  photostats  and  develop an  equation  to  scale it  on  the  chemotactic  index. So  you  can  see a  much  higher  chemo  typic  index for  the  might  than  the  dark. So  just  to  say  like  further  away  from the  one  is  a  more  intense chemotactic  response. So  just  a  way  to  quantify that  you  are  seeing what  we're  seeing  visually. The  next  result  I'd  like  to show  you  is  a  little  interesting. Now  we  want  to  compare  step  back  a little  bit  and  compare phototaxis  and  chemotaxis. And  then  one  to  4.4  533. We  did  these  heat  maps  basically  of the  different  photostats  that you  see  that  coordinate  with  each. We  saw  some  gradual  movement  and  phototaxis. We've  seen  a  really  cool  thing  of  4533 and  that  some  positive  negative. But  what  we  really  focused  on here  is  the  first  instance. Started  seeing  some  collective  migration. So  if  you  see  where  the orange  and  yellow  colds  are, it  almost  looks  like  at  3  H  and  6  h  in  both. They  almost  wanted  to collect  in  the  center  of the  dish  before  they  decide to  move  along  the  chemical  Brady. So  that's  what  we  were  more interested  in  to  investigate. And  then  we  also  considered the  fact  that  photothtechnic  response, which  you  can't  really  eliminate phototaxis  in  these  experiments. We  wanted  to  eliminate  type of  directional  device  that might  have  caused  them  to collect  but  maybe  it's  not  chemical. Brad,  again,  we  did  this  in  C124,  CCC  4533. So  the  top  row  it  goes  by, top  second  row  goes  by  left, third  les  boys,  right? So  you  can  see  in  all  of  these  in  CC  14, they  collect  in  the  middle first  and  then  decide to  respond  to  that  chemical gradient  in  all  directions. But  before  533  we  saw  something  different. So  we  didn't  see  this  response  at  6 H.  There's  really  spread  out. Within  12  H,  we  start  to  see circles  that  I  drew  that there's  a  little  more  intensity  there. But  eventually  they  do  respond positively  to  the  chemical  radiant. But  they  don't  collect  like  CC  one  to four  you  had  who  blocks  that?  A  one  gene. So  we  are  determining that  CC  one  God has  enhanced  collective  migration, which  in  turn  enhances stability  to  Mosconemonia. Then  we  wanted  to  investigate  the  role  that this  ad  one  gene  or protein  was  playing  in  chemotaxis. That's  when  we  obtained the  transgenic  strains, number  60,  number  13. And  that's  confirmed  by  Westok  that they  have  the  presence  of the  recent  at  one  gene. Confirmed  that  phototaic  activity  which C14  sifegative  photota  response  613  dot. Then  we  looked  at  the  chemotactic  activity. So  these  are  sons times  that  I  mentioned  earlier, roses  to  control  no  chemical  gradient, but  when  we  have  a  chemical  gradient, we  see  that  response. The  aggregated  response  is  CC  one to  four  but  not  so  much in  6.13  which  again they  have  that  recombinant  add  one  gene. So  we  are  determining  that  add one  is  really  playing a  role  in  collected  migration. And  also  wants  to  show  you  this  cool  video. We  basically,  so  one  of  our  pet  assets, put  it  under  the  microscope, under  chemical  gradient. So  to  the  left  is  no  matric, to  the  right  is  excess  matrogen. This  is  what  we  saw, 01  of  those  aggregated  cells. If  you  could  see  that the  individual  cells  are almost  pushing  this  aggregate, so  this  is  where  this  paper  stop. But  just  to  allude  that there's  a  little  bit  more complexity  to  chemotaxis, Chemotactic  than  we  probably  thought. So  what  kind  of  connections, what  kind  of  communication  is  going  on  as these  individual  cells  push this  group  of  cells? So  let  me  speak  more  into the  complexity  of  the  collective  migration. We  also  wanted  to  make  sure  that  the aggregates  that  were  formed during  elective  migration, we  send  the  microscope  soft  stress. You  just  did  a  quick  experiment induce  soft  stress  and  you  can see  that  these  are a  collection  of  much  less  cells. And  they  form  a  membrane  just  to show  that  is  different than  what  we  see  during  collective  migration. So  it's  just  not a  strict  response  actually  is collective  migration  and  also with  the  independent  workings  of  light. And  one  we  proposed  this  model again  speaking to  more  of  the  complexity  of  it. As  light  is  present, it  induces  more  of  collective  cell  migration, which  induces  a  better positive  chemotactic  response. And  with  the  lack  of  that  one  gene, it  leans  more  towards  single  cell  migration and  still  can  produce a  positive  chemotactic  response, but  not  so  collective. So  just  to  say that  this  chemotactic  mechanism probably  has  more  aspects  than maybe  even  the  photo  tactic  mechanism. And  there  is  a  lot  of  literature that  needs  to  be  published  on  this. So  I  just  recently  published  my  paper. I'm  very  excited  about  that. Just  to  conclude  that the  migration  patterns  of Ptas  and  chemotaxis  are  different. And  I  might  say  that  chemotaxis might  be  a  little  more  interesting than  taxis, and  that  ammonium  chemotaxis at  least  is  more  efficient with  collective  migration. And  that  collective  migration can  be  enhanced  by, and  weakened  by  add  one. And  that  the  mechanism  of  chemotaxis is  probably  more  complex  than  we  think, because  there's  specialization  with aggregated  cells  and  aggregated  cells. And  I  just  want  to  acknowledge the  team  who  helped  me  publish  my  paper, my  PI,  Dr.  to  my  Alexis  string  to  help  me. And  she  gave  a  talk  earlier, Dr.  Walker  and  I sue  special  transgenic  strains and  then  Dr.  Melvin  and  a  Dr. Elihu  who  helped  us  with  a  lot  of  the  model. And  I  would  take  any  questions. So  have  you  talked about  this  relationship  between  light, carbon  and  nitrogen  tactic  movement? Is  there  a  reason  why carbon  designation  light  and nitrogen's  differently  link  between the  two  that  you  can  in  the  ecological? So  actually  I  mean I  believe  so  but  actually  that's something  that  perplexes perplexes  because  like  if  you  think about  down  migration  when  they're  more active  at  different  times, whether  they're  in  light  or  dark, our  experience  really  matches  that. So  I  think  this  speaks more  into  the  complexity  too, or  maybe  the  way  that  we're doing  our  experience. A  definite  link. But  the  explanation  for that  link  that  was  very, very  beautiful,  I  was  really  intrigued  with some  C14  movies  were making  a  control  condition, You  have  no  Miu  gradient, you  still  want  them  collection. And  I  don't  know  what  you  think  is happening  there.  Yeah,  right. There  was  no  chemotactic. And  two,  I  don't  know  if  this is  like  a  question  or the  looks  a  lot  like  the  collectibles. Dsc  want  literature,  that  may  be an  interesting  place  to  look  at the  dictature  what  we  see  in  CC  one. Even  when  we  place  the  moncler, they  will  still  aggregate. I  think  that's  due  to  the  lack  of  the  egg, but  just  based  on  the  way  I experienced  in  photography  at the  top  and  I  think  that photo  technic  response  is  very  strong, very  real,  and  what  we  do,  so  all  we  try  to, because  no  matter  what,  we're  going  to get  a  photopic  response. So  all  we're  just  trying  to  do  is place  them  directly  under the  light  to  minimize them  going  left  or  right, because  they're  going  to go  in  that  direction. We  can't  keep  the  homogenous  at  all back  here  on the  right  cure. The  light  enhancement  of phototaxis  was  dependent  on  photos. Dependent  on  that  is an  enhancement  of  chemotaxis. Yeah.  Is  that  synthesis? It's  very  possible.  But  I  will  say  we  did an  experiment  where  we switched  our  lightning  day, light  and  dark  day  cycles. And  we've  done  per,  in  their  day  cycle  and then  their  night  cycle and  switched  their  drawing  cycles. And  it  hasn't  changed  none  of  our  results. So  you  would  think  it's  involved. But  what  I've  been  seeing, based  on  the  experiments we've  done,  maybe  not. Thank  you  very  much. Want  chemotaxis? Yeah,  I  mean,  that's  something  to  look  at because  that  experiment  that  we  did  just, we  actually  did  it  for  the  reviewers, but  we  didn't  put  it  in  the  chemical  rate, so  that'd  be  a  good  thing  to  try. Hello,  thanks,  nice  stock.  I'm  here. As  far  as  I  understood, in  the  time  zero, you  get  both  the  Ccd and  the  moving  and  it  both  evolve  in  time. And  it  seems  to  me quite  hard  to interpret  the  collective  movement. I  wonder  if  it's  possible to  the  technical  readings  and  let  it  to  reach a  pseudo  stationary  state  so  that  you  have and  then  you  respond  with  the  cells  in the  middle  and  then  you see  which  way  it  goes. And  then  you  can  discriminate between  the  diffusion  of  the  cells itself  and  any  other  type  of movement  that  can  come  from  pure  phototaxis. I'll  tell  you  a  few  things  we  have  done experience  where  I  place the  chemical  gradient  and then  I  pipet  the  algae directly  in  the  center  and  I  think  it  moves. It  actually  responds  fast. Because  there  I  think  because  there already  and  I  also  tell you  I  have  the  same  idea that  I  want  to  let  the  gradient establish  a  little  bit  for  a  few  hours, but  then  there,  there  will  be  no  way  for me  to  homogenously  introduce  the  population. Right?  Because  then  I  would  have to  disturb  the  chemical  gradient. That  is  a  challenge  that  we  have. Once  you  have,  if  you  have modeled  the  concentration  across  the  dish, you  could  give  spike  cells in  the  concentration  that corresponds  to  the  concentration  of the  exact  position  where  you're  spiking. Yeah. Then  there  wouldn't  be  any  breeding  time. Zero. And  by  the  cells. Yes.  So  you  showed  us a  novel  Ceding  event during  cable  Texas  and  I'm  wondering because  cell  aggregate  before  in  the  video that  they  migrate  was  the  ammonia. So  I'm  wondering  whether  it  is  you  can  try some  cell  adhesion  molecules  mutant. Or  you  would  look  at the  flagella  behavior  when the  cell  the  at  that  blow  side. Very  interesting. We  have  looked  at  some  blop  earlier. We  looked  at  because  we  can  induce whether  they  form  of  the  surface  or  virus. We  try  that  the  photo  perfect. We  didn't  from  the  top  right. So  we  did  this  experiment with  light  from  the  bottom to  make  the  Navy phototaxis  one  at  the  surface. I  didn't  see  any  difference. And  so  I'll  suggest  then is  to  try  to  move  lights. And  I  didn't  experience,  they  didn't  move  at all  and  I  discovered  that  we  have  Pogo. So  that's  also  a  challenge. Finally  effects  to  taxis  there. Anybody  knows  of  any  lights that  have  Pogo?  Please. Yeah. I  also  very  much  like  the  time  moves  for phototaxis  photo  keeps  that  foot  in  the  dark. We  try  to  nitrogen  star  says  prices because  of  course  wait. I  want  tried  to incubate  nitrogen  three  media  to  see. No,  we  haven't  tried  that. So  I  immediately  take  them  and  put  them international  media  and  tried  to almost  immediately  start  by  experience. So  no  period  of  starvation, I  will  say  I  haven't  done, but  Dr.  Milton  did  try  something  like  that and  didn't  seem  to  have  much  difference. But  I  haven't  done  it  in  my  success. Have  you  looked  at  nitrates? Have  you  looked  at  nitrate? That  the  possible  and  also  I've  looked  at sales  which  would  be  needs to  be  able  to  move  nitrate. Vi  haven't  looked  at  either, but  I  did  look  up  some  literature. Somebody  had  the  same  question  before. The  literature  that  exists has  only  been  done  in  ammonium  nitrate. Not,  no.  So  maybe  there's something  at  the  charge  there's  no favored  something  on  the  charge. I  haven't.  Thank  you. My  name  is,  my  name  is  and  Janet  favorites. These  are  dings. One  such  arm  is harder  which  is  compared  in  all  uterus  of course  including  normally  the  micro and  this  effector  protein out  a  forms  a  complex  called Sc  and  indicate  that's a  partially  complete  sip My  the  risk  then  decree bound  or  inhibit  the  transpression  wizard, resulting  in  negative  dilation or  target  expression. The  cloning  has  three  as  for which  the  Pos  has  been  identified as  the  most  equals  M  instance, similar  micro  have  identical for  example  in  As  also  these are  clearly  that  the  micros be  seen  in  context. The  Asps  and  print deficient  in  micros  does  not show  model great  comparable  micros. so  in  this  show  that they  microbes the transfionosrurigyptyssimicutansusingvibsoluecjustwmicroy looking  at  the  party  of micro  we  can  get a  clue  micro  by  identifying  the  ts And  the  Charlie  that  opined  Chsld, we  can  see  the  high  frequency  of the  single  nucleotide  period. So  the  opinion  and the  notes  wans  and perimeters  were  grown  in  growing  frequent, Different,  especially  in the  half  of  the  light  period. And  besides  a,  an  other  gardens  extinguished. Mark  also  show  the  decrease  in the  variable  cast  proteins  at  the  8  H  of the  Tics  or  unchanged in  compared  with  strength  that  microns. The  transients  unlike  most  do  microstone with  a  sequence  cut  complement  micro  is efficient  that  Sidle  be  depicted  as  Y, the  sped and  also  binding  benefit. So  the  dedicated  also  included Fst  division  and  Fain state  conditions  to  include  that  rapists in  provision  which  is the  heat  method  and  the  success then  identify  similar  target  chains and  this  is  the  heat  slip is  one  to  applies  to  the  other. Wins  over  100  engines were  involved  in  the  Gina. And  how  did  functioning development  then  transport, localization  regulations and  phase  regulation  disability. Only  a  few  mining in  have  been  started  in  India, but  recently  as  can be  moved  into  the  and  chain  using the  system  actually  successfully introduced  the  three  timing  into  the  Oga. Different  theoretically  method  can be  applied  to  other  mining  voting. If  you're  interested  in  we  can  open question. Yes. Yeah,  so  the  same  article, three  that  overaclatesptscscs  is  that which  is  both  of  them  control  same  arts, would  you  be  able  to  be  present  within  the next  also  the  targeted by  the  same  micro  Let  me  the agreed  And  both  cast  then  for  the  drop, Consolidated has  over  100, 200  microbes  deficit. You  use  data  to identify  micro  targets  overall. Can  you  make  links  between one  particular  target  and a  particular  microdata? We  have  no  link  between  T,  Tiger  and  Micro. Just  I  was  wondering whether  it  would  be  possible to  the  Roy  to the  target  or  before  you  make  the  library. Invention  Micro  is  not  shown  at  this  point. We  can  micro  and  targets, we  can  find  a  combination  of  micro. And  that  is  difficult. The  expansion  of  stable  level. So  I'm  wondering  what  is  logically relevant  in  the  microlevel. I'm  quite  stable  over the  geroge  relevant  micro. Next,  find  a  reason why  I'm  going  to  have  to  chain. Thank  you. Next  will.  Okay. Good  afternoon  everyone.  My  name  is German  from  University  of  Illinois. Please  here. And  to  have  a  chance  to  give my  talk  in  this  session  today  I'm going  to  talk  about  my  research  focusing  on the  application  of  tool for  the  achieve  patient  specific. Luckily  I'm  one  of the  members  who  developed Cyp  in  terms  of  a  system. So  at  the  time  we  just  focus  on the  crystal  because  we want  to  stay  go  like  that. But  after  that  we  try  to  use  crystal. So  it  is  not  plot  P  using it  and  dividing  Act  integrates  so  we can  increase  the  intricacy  in the  frequency  up  to  36%  We  publish the  theta  here  and  then  we  already  published all  power  in  2017 after  I'm  using  this  Crisps tool  and  I  realize  that show  very  interesting  pattern which  is  the  Gen  Act. There's  no  Les  percent  case, there  is  no  divisional  scales. I  define  and  classified, there's  a  repair  of the  cassette  type  is  scales. I  analyzed  all  of  my  Cat  YG, and  then  we  found  that  type  almost  14, 23%  of  the  button  shows  even  though  ****. When  I  see  this  feature, one  idea  I  have  is  that  if  by our  Crispr  target  is this  area  start  near the  start  of  the  ambiguous  in, and  if  we  introduce  this  case, which  is  a  spin  Barker, and  there's  a  constructive  stronger  here, mix  them  together  and  we  collect  the  mutant. We  can  by  C  replace the  original  Romer  and then  introduce  this  target in  using  our  Strop  constant  Romero to  move  Sp  target  chain. Because  of  the  physical  case, it's  not  difficult  to  make conduct  the  non  over expression  of  the  expression more  difficult  because  of  this, a  reason  there's  a  gust, there's  a  fictional  effect, something  like  that. Recently  I  mechanism, interest  mechanism  is  discovered by  the  large  closed  group. And  then  they  realize  that when  we  introduce  the  NA, of  the  target  of interest,  it's  not  expressed. But  if  we  introduce  the  interim, the  CNA,  you  can see  the  increase  of  the  mint. Actually  I  also  tried  to  apply  to my  Tara's  not  easy  because sequence  DN  as  cloning and  come  size  is  very  big in  intro  and  side is  getting  increased  whether they  introduce  the  CDNANANI  do. introduce  prom  just  using their  own  system  system  so  we  can  increase the  expression  and  also  we  can  avoid this  functionally  problem  olin  method we  set  up  this  method we  kick  the  targeting  of  course  our targeting  was  one  because  b  is  very  wet  and then  a  few  times very  strong  with this  one  and  then  this  is  the  p We  can  follow  the  Asp  using. In  this  case,  our  target is  not  the  promoters, is  the  three  meteors in  front  of  the  sytem  extension. We  design  another  version  of  the  Dodd. There  is  the  stuff  here, and  the  three  prime, and  this  part  from  the  Pm  resistance. We  introduced  this  one to  remove  the  sytem  extension. And  also  we  tried  to  overspread  Pj, which  is  the  off  of  the  ****. This  is  the  color  of  the  mutants, so  this  is  the  Opic  expression  and  this indicates  used  by  locking. And  this  is  the  further  further the  Ptp  and  remove  the  C  from  their, we  can  compound  compound  the  Ocp  of  Pps  by  C. Then  in  this  pane that  compared  to  the  data  can  see  getty. Then  the  Act  increased in  this  ph  B  word,  Pkc. This  is  a  summary  of  the  mine  study. Usually  we  use  nine  tools  as  a  tool  for  out. But  we  can  use  this  lack  in  to increase  the  expression  and  work modulate  of the  expression  by  system  modification. And  also  this  method  could be  avoid  this  kind  of  problem. Presale  Cdos, also  just  modification  tool. We  can  we  can  use  the  tools and  protein  trafficking  or authorization  and  prohibiting  et  cetera, so  it  has  a  lot  of  potential  it. I  work  for  the  right  project  and  pointing  and our  our  project  supported by  about  this.  Thank  you. And  I  just  very, very  quickly  question  was  the  efficiency? Individuals  information  was  the  efficiency. Yeah,  our  packaging  issue,  focus  percent. I  was  wondering  if  anyone  have  ever tried  different  homologs  because they  developed  paper  within, for  example,  being  active, more  active  at  lower  temperatures. Has  anyone  ever  tried  using  different  logs? You  also  tried  to  put you  have  tried  to  also  a  small  epogen, Why  you  overcos? Yeah. Actually  there's  time  in  my  Well, I  introduced  having  Yeah, because  that  protein  is side  speak  is  membrane  proteins. We  tried  to  try  time  to  make anbs  that  I  take  the  prom, I  take  the  Hp behind  the  protoceworkyknyquestsks and  and  you  know, there's  a  maximum  of  the  sciences. Yes.  Yeah.  Usually  is  under two  Kd  because  of  A  and the  side  is  not survey  because  of  our  Homer  paper. So,  in  this  paper, I  think  in  this  paper we  introduce  the  yay. Last  question. I  think  Kt  because  you're like  you  still  with  you  As  seen. You  also  like  compared  production. Production. Yeah,  the  paper  you  would  like comparing  and  the  productions  paper, which  method  is  useful  to  get bent  was  the  Nv compared  to  the  conventional  method to  our  compter  chase. We  introduced  the  Pv  promotes  nav  sequence  to the  inotypenparevvjhowreotype introduced  to  then. Thank  you  and  we want  to be at  all, right? Is  the  same  words,  kind  colleagues  have accused  me  of  wanting  to  have the  last  word  and  I  guess  the  program,  right. I  know  that  the  dinners waiting  and  people  are probably  getting  angry, but  I  won't  be  considering  our  12  min. I  think  this  is  something  that  most last  year  could  take  away  something. And  it's  a  demand  that  overarch, shift  everything  from photosynthesis  metabolics. It's  something  that  everyone  needs  a  bit. And  what  I'm  going  to  talk about  mutagenesis  with  risher, I  don't  have  a  fancy  name  I just  told  is  making  mutans. I  have  over  the  last  three  years  or  so, it  before  before  made about  60  different  lines in  many  different  backgrounds. Sometimes  double  tax,  triple  tax,  um, about  half  of  them  just plastic  lockouts  and  they're  in  the  top. And  walk  you  through  from very  simple  designs  to  very advanced  and  very  brief  form designs  that  I've  recently  been  playing  with. What  I  understand  or  what I  call  precision  genomic  is  that  in traditional  pmoeneticslymse has  double  from  break. It  likes  to  integrate  st and  it  does  that  fairly heavily  And  that  leads  to astonishing  resources  like  lighter. Convince  anyone  on  my  stats  orientation of  these  insert  is  pretty frees  the  place  where it  integrates  and  attributed. That  makes  a  quick  library  but  it's  also a  hindrance  when  you  want  to  do  editing. Instead  we  know  that the  cast  nine  can  bargain  that  break. We  can  also  construct cassettes  that  have  homology feeder  side  that  will just  neatly  integrate  with the  directionality  that works  pretty  well  how  we  construct the  donor  Dna  for  a  classical, I  call  the  knockout, that's  probably  a  better  word. The  gene  disruptions,  machine disruptions  that  we  select the  double  strand  brake somewhere  on  the  second  Xor. And  then  we  will  insert  a  cassette like  this,  analyzing  the  system. And  then  in  the  course  of  this  work, I've  also  flown  a  set  of four  different  very nice  resistance  markers  and all  the  same  framework  cassettes. And  what  we  do  is  we  would  take the  homology  50  basic pairs  upstream  and  downstream, the  same  part  of  the  grid, George  Woodman's  left, and  we  make  linkers  that tax  as  this  homology  here. Importantly,  there's  a  little two  s  restriction  sites all  on  those  linkers. And  we  complement  those  linkers divisionally  with  homologous  regions. The  case  and  I  do  everything by  homology  works  very  well. By  this  way,  we  can  plasma  verify  them. And  then  once  done, we  can  cut  this  case  out  and  insert. It  doesn't  take  a  lot  of  creativity to  realize  that  if  this  works  well, you  can  also  make  fluorescent  fusions, for  example,  by  choosing a  double  strand  break  that  end  up  this  gene. And  putting  one  end of  the  homology  on  the  side  of  this said  and  the  other  end starting  complemented with  the  neon  green  tag, for  example,  epling  three  prime  TR. And  then  turn  saying  that  you  put  that  in, that  works  really  well  how I  transform  these  cells. It,  the  cell  walls  with  talsey  heat  chop them  and  then  electrodes  ming  with  the  star. Dnai  use  neon  electroporation  system, which  lets  you  have  just about  ten  microliter  cells. It's  really  nice,  very  small  volume say  resources  that  way. That's  typically  first  hand micro  reaction  results in  a  plate  like  this  with  about  500  colonies. This  you  don't  need  more than  than  12  of  these  reactions for  in  then  I  think  colonies, I  screen  them  until  I  have  Flores. Now  I  want  to  highlight  maybe two  very  important  things  in  this  process, the  donor  DNA  quality  and  also  the  Atos. And  want  to  give  you  a  system  numbers because  the  kit  that  you  used  to do  this  thing,  medical  law, if  you  prepare  last  two  different  create  kits which  medicinally  are  already  pretty clean  and  you  get  when  you  get  the  results and  you  look  at  narrow  drop  readings  that give  you  the  quality  of what  you  expected  there, you  would  assume  either almost  the  same  quality. There's  hardly  any  difference in  the  inspectors  topic  readings. The  thing  is  that  the  spectroscopy in  this  case  can  lie  to  you  a  lot. And  when  you  look  at  the  fluorometricessay, which  is  known  to  be  much more  precise  in  measuring the  reported  vehicles  and  DNA  in  this  you notice  that  the  ratios of  what  you  actually  have, what  you  thought  you  are quite  different  between  kids. And  indeed  when  you  transform  this and  use  the  good with  nothing  you  get very  little  colonies  is  well  known. Soliz  breaking  you  have to  give  you  roughly  the  same  amount  of colonies  but  when  you  look at  how  many  of  them  are targeted  to  the  right  spot, it  really  matters  which  one  you  use  and that's  I  can't  say you  get  rid  of  those  changes. I'm  going  to  show  one  of those  examples  where  the  number  tag, this  fact  56  ciliary  tapping. And  when  you  have  pre  selected a  few  mutants  where  you  already  know that  the  nimologyjoint  amplify this  entire  insert  up  to the  resistance,  the  diffuse. What  you  notice  is  not  all of  them  have  the  accepted  correct  size. Some  bands  have  a  different  size. The  sequence,  a  whole  bunch  of  those  as  well. And  what  you  can  notice,  visualize  it  would be  made  like  rearrangement  blots. That  pins  will  absolutely, during  integration, exchange  fragments  of  your  cassette with  parts  that  are flying  around  and  sing  patterns. They  are  micro homology  driven  fragment  exchange. But  everything  here  that is  looking  like  it  has the  correct  side  and  usually  you  can  already tell  by  that  even without  sequencing  is  always. Usually  in  a  normal  experiment, between  25,  30%  of those  colonies  with  this  protocol will  absolutely  look  correct. Also  put  in  and  if  P  marked coheres  Celia  next  to these  wonderful  little  blow  tips like  a  ray  showing, waving  around  the  low  stick at  the  end  of  the  very  huge. Now,  the  screening  is  very  important  because once  you  start  making  mutants  in  a  bit of  a  more  hydrate  fashion, you  get  a  lot  of  them  much  as  have one  or  two  mutants  lot.  So  you  get  a  lot. I  spend  quite  some  time  finding  good  mixtures for  PCR  that  are  also  able  to  work. Acr,  you  don't  have  to  purify  DNA, you  just  use  cells or  heat  live  cells  with  template. This  works  reliably,  even  though 384  well  plays  in  three  microled  reactions. But  is  it  the  nice?  Because  you  can  do a  lot  of  that  and  it's not  very  expensive  this  way. This  is  all  the  works  with  PCR  dice. I  do  almost  all  there  screening  on APCR  as  opposed  to  a  traditional  PCR. Because  it  lets  you  do  things like  directly  determine the  melting  mating  type just  by  melting  products. Now  this  lets  you  do  most  of the  designs  you  want  to  do  in your  daily  work  use  term. You  can  choose  thanks  to  your  terminus. And  that's  already  nice. But  what's  nasty  is to  have  this  resistant  cast  always  used. It  helps  because  of  course, it  up  the  efficiency  quite  a  bit. But  since  we  can  also  screen  now. Well,  I  tried  something  else  where  I took  the  C  terminus  of  this  it  40  16. I  chose  a  cut  side  closer  to the  end  and  I  instructed  a  cassette that's  just  the  linker  and  a  fluorescent Protein  Plomo  arms  and transforming  that  is  not  exactly  terribly efficient  because  you  have  side  force  here. You  have  to  hold  that, you  find  one  that's  correct or  what  you  can  also do  is  you  can  choose  yourself  a  second  gene, somewhere  like  way  from  another  chromosome. And  you  can  target a  classical  insertional  exertion  cassette to  that  second  part. And  this  is  borrowing the  traditional  transformation  approach for  making  insertional  mutants like  it's  pretty  standard  for  a  long  time. That  totally  works  of  course, because  it  doesn't  work  as  well. So  the  primary  efficiency goes  down  to  a  few  percent, but  since  you  can  screen  plate, it's  not  that  much  of  a  problem. Then  you  make  games,  you  hatch  them, fax  machine  just  to  efficiently  signal, to  make  isolate  them  into  Sable  wells  plate. And  you  can  see  the  result of  such  crossing  here, the  original  Paradise  if  Tv  46  and scarlet  strain  has  shorter  flagella but  of  course  sent signal  and  once  you  press  it back  out,  everything  returns  well. The  cells  are  no  longer  resist  Ugt. I  want  to  end  this with  a  more  advanced  design which  is  I  chose alpha  tubulin  for  an  internal  Ad. And  why  is  alpha  tubulin  difficult? Is  because  we  have  two  copies of  alpha  tubulin  momon. If  you  want  to  insert  anything  here  on the  terminus  is  very difficult  to  find  a  unique  guide. Because  the  guide  that  is  anywhere close  to  here  will  both  of  those  genes. And  that's  really  not  what  you  want. What  I  did  instead is  you  can  see  it  around  here. There's  regions  where  you  can  find guides  that  are  obviously unique  because  tugs  don't  align. And  I  chose  two  Soci  use  in  total, the  three  Orp  and the  cassette  that  looks  something  like  this, that  you  have  the  homologous  region. With  the  remainder  of  this  plus  UTR, you  put  in  the  nonres, some  introns  and  you complement  the  whole  thing at  the  end  of  the  first, the  hydro  first  and  the  remaining  parts and  of  course  this  can  then replaced  that  little  part  that you  come  out  perfectly. And  of  course  the  same  way,  again, Rs  four  in  the  as  the  selection  marker. I  have  to  say  it  didn't  work  as well  as  I  hope,  but  it  didn't  work. You  do  get  fluorescent  two  through  and I'm  not  sure  if  it  was the  entrance  that  are  not  very  nice. But  just  attack  that, you  do  have  attack  there even  within  the  current  edits. You  have  things  that  you  need to  consider  past  just  it. And  with  that  I  want  to  thank my  three  laps  as  well. The  very  nice  support that  we  have  a  the  Moscow  Society  with many  facilities  that  help  us red  a  lot  of the  day  to  day  routine  of  our  hands, we  can  focus  on  more  interesting  things that  I  would  I  guess, like  to  finish  or  close  the  conference. And  here so  you  look  at a  similar  pens,  the  cytosolic  incubus. Yeah. Ar  Carl  has  a  really  nice  Sfpp or  integrated  tubulin  strain. I  kind  of  started  even  remaking a  few  strains  that  we  already  have. Technically  with  the  old  insert  method, surely  because  crossing notions  tags  gets  way, way  easier,  especially  once  you start  stacking  them  on  top  of  each  other. Because  of  course,  normally  you would  have  one  in  four  chance and  then  suddenly  you  get  a  one  in two  chance  cross.  Correct? I  see  that  previously because  tubulins  very  complete. This  one  still  have  Tslp  two  undisrupted, but  it  has  the  alpha  tubulin  one  tag. I  have  not  knocked  out alpha  tubulin  two  yet. I  don't  know  what  happens. It  may  die. The  you said  the  G  Rs  are  each  basis  super, super,  super  short  at  that  short. And  is  there  any  reason  to speed  that  the  road  work  better? Well,  I'll  be  honest with  you,  on  average  unit. So  George  Whitman  did  some  work  there. He  already  found  that  50  is  usually, I  think  he  went  up  to  100, and  they  didn't  see  so  much  of  a  difference. And  50  is  just  a  nice  size. It's  easy  to  get  a  fragments  that  are  small and  honestly  the  efficiencies of  integration  are  so  good. Why  would  you  need  a  different? Thank  you  for  the  great  talk here  in  West  side. You  mentioned  I  need  two  keys, Da  and  transformation  efficiency. Are  you  saying  different  keys? You  may  have  a  different  contamination. The  affect  adding  because the  ends  of  the  DA  get  truncated. That  affect  equilat  enzymes  and  then purify  must  be  something  in  there  because  you see  the  fluoro  genic  acid  just  gives  you more  DNA  read  out in  one  case  and  not  in  the  other. There  must  be  something  in  those  samples. It's,  I'm  pretty  sure  it's  up  to  the  DNA. It's  not  a  problem  when you  do  insertional  work. Traditional  for  the  mojo doesn't  seem  to  be  effective, but  the  homology  directed definitely  seems  to  be  effective. Thank  you  again, in  the  Tua, it  appears  that  you  put  set  into  the  UTR, isn't  there  an  Asp  which  will  prevent expansion  and  the  tubulin is  N  terminal,  not  AC  terminal. And  yes, it's  an  terminal  tag  because  I  didn't  want  to put  anything  on  the  C  terminal tubulin  that  would  give, it  inhibits  into  a  certain  things like  disorder  and  multiplies. It's  kind  of  another  place where  you  want  to  put  a  tag. So  I  put  it  in  the  inter, I  missed,  I  put  it  in.