And  then  we  will  have  a  discussion. We'll  have  actually  the program  multimedia  work  library and  then  we  have  a  Musgrave. Got  this? Yes,  Tray  Smu. And  he's  going  to  introduce  himself  where? Yeah. So,  my  name  is  Travis  Smm, a  senior  scientist,  Algae  innovations. Today,  I  just  want  to  give  you  an  overview  of our  company  and  what we're  doing,  our  capabilities. I'll  be  talking  about  how  you're moving  clammy  from a  lab  organism  into  industry and  dynam  first  off, what  makes  clammy  attractive  for  industry? Normally  you  think  about  it  as a  model  organism  in  the  lab, not  so  much  industry  but  like E's  algae  and  E.  Coli, it  had  a  huge  knowledge  base  and that  you  think  is  a  good  reason  to  use  it. You  have  strain  diversity transformation  ability. Other  things  you've  heard  about, protein  folding, mating,  Some  other  things  you  might not  be  aware  of  is  that  it's  fermentable. We  have  some  clinical  data I'll  talk  about  in  a  minute. The  cell  wall  has  a  benefit  because it's  not  as  robust  as  some  other  ones. Nutrition  that  I'll  talk  about. And  then  there's  a  lot  of  phytochemicals  that other  fermentable  organisms  just  can't  make. The  way  trichlogy  came about  was  about  ten  years  ago. It  came  out  of  Stephen Mayfield's  lab  at  UCSD. The  original  point  was  to  make therapeutic  proteins  in  Flaming, and  then  we  haven't  delivered in  a  whole  sale  matrix. And  this  necessitated  safety  testing for  the  biomass  in  animals  and humans  along  those  lines to  prove  it  was  safe. You  can't  just  say  it's  safe. You  got  to  do  a  little  bit  more  work than  that,  especially  in  industry. We  went  through,  sorry, went  through  a  whole  bunch  of lamia,  did  all  this  testing. We  did  get  approved  for  grass  in  the  United, in  the  United  States,  both  by an  independent  conclusion  and  FDA. We  have  grass  for clama  now  in  China  and  Singapore, and  we're  working  on  that  in  Europe. And  then  we  also  have other  strains  that  are  either  in the  pipeline  or  already approved  for  the  grass  status. But  beyond  that,  I just  proving  that  it  was  safe. We  also  saw  some  benefits  in  mice, pigs  and  humans  that actually  showed  a  gut  health  benefit. And  this  was  without  the  recombinant  proteins that  I  was  talking  about  before. This  is  another  factor  that led  us  to  get  away  from the  recombinant  proteins  and  just  focus  on the  whole  cell  biomass  and  how it  could  be  used  as  a  food. Today  we  have  five  departments. Starts  with  the  strain  development, which  finds  new  strains and  develops  new  strains. Process  development, which  takes  those  strains and  screens  them  for  fermentation  ability. And  then  figures  out  the  scale  up. We  have  food  science  department  that takes  that  biomass  and creates  new  foods  with  it. And  then  sales  marketing, which  of  course  moves  it  to  get  to  consumers. And  then  regulatory  affairs which  make  sure  everything  is  safe. Just  an  example. At  the  top  of  the  strain  development  team, we  started  off  in  this  case to  get  our  Ta  114  strain. We  started  off  with a  green  strain  through  a  combination  of UV  uto  genesis  facts  and  mating. We  came  to  this  red  strain. The  red  strain  creates  poppy nine  above  5%  of  biomass. That's  a  biosynthesis  before  low  chlorophyll. We  do  have  independent  conclusion of  grass  status on  this  and  we  think  this  is  food, a  media  flavor,  so  it'll be  exciting  fluid  green. But  along  those  lines,  getting an  idea  for  a  phenotype is  just  the  beginning. We  can't  just  get  one  strain with  the  phenotype  we're  looking  for. We've  got  to  do  a  little  bit  more  than  that, because  we  can't  grow it,  it's  useless  Industrially, we  go  through  this  long  process, all  the  way  down  to  fermentation, from  flasks  to  fermentation, to  make  sure  that  we  can  grow  it a  scale  just  green. And  the  red  strain,  we  have  hundreds  of strains  now  in  our  library. These  all  have  a  unique  phenotype. And  they've  been  screened  for the  ability  to  go  into fermentation  and  produce  at the  levels  we  need. Normally,  you  think  about  clammy and  algae  and  you're  thinking about  phototrophic  growth. Why  are  we  looking  at  fermentation? Well,  actually  not  needing light  is  a  huge  benefit  because  it  lets  us get  to  those  higher  densities that  you  really  need  to  get cost  effective  for  industry. There's  a  lot  of companies  that  try  to  do  it  in  ponds  and such  and  most  of them  that  haven't  made  it  today. It's  also  sterile,  so  you don't  have  to  worry  about  contain, you  do  worry  about  contamination, but  most  of  the  time  it's  good, is  really  ideal  for  a  food  product. Then  you  also  have  tightly controlled  parameters  that  really helps  with  the  scale  and the  optimization  of  your  growth. This  is  what  our  capacity  At some  examples  we  have  over  600  m  in  house. Starts  off  with  our  amber  system which  are  bank  of 24  automated  bioreactors  That  allows  us to  try  a  lot of  growth  conditions  and  stuff  in  AE  fashion. And  starts  off  with  on  our  fermentation, we  then  go  through  a  variety  of different  capacity  up  to  150  liters. And  we've  got  some  downstream  options  too. So  this  allows  us  to  get everything  on  a  small  scale. That  way  we  can  scale  up  to what  we're  really  need  for  industrial. With  all  that,  we've  really  vastly improved  the  productivity  of planning  back  in  the  day. Maybe  get  a  couple  of  grams  per  liter. Recent  reports  have  up  to  25  g  per  liter. We  can  get  up  to  over  150  g per  liter  in  our  fermenters. And  what  that  has  allowed  us  to with  this  red  string say  it  didn't  grow  all  that  well. So  we've  optimized  it  all  the  way  from flasks  into  small  fermenters,  big  fermenters. And  that's  allowed  us  to  then go  to  our  CMO  with optimized  proven  manufacturing  protocol. And  they've  grown  at  180,000  liters  scale. And  if  we  went  full  war  with  this, we  could  get  up  to  5,000 metric  tons  per  year. And  that's  really  the  kind  of scale  you  need  to  have a  commodity  food  products  that can  be  competitive  in  the market  we're  talking  about. Food  plant  is  also has  a  lot  of  benefits  nutritionized. We  can  get  the  protein  up  to  50%  It has  all  the  amino  acid, essentially  amino  acids. We  have  oils,  and  vitamins and  minerals  that  we think  made  a  good  food  product. And  then  I  said  we have  a  poscience  department, they  made  a  variety  of  foods  with. This  is  alternative  tuna  that actually  I'm  not  a  vegetarian, but  it  was  pretty  good. If  you  put  it  in  tuna  salad, you  really  couldn't  tell  the  difference. They've  made  a  variety  of  drinks,  meatballs, snacks  and  desserts and  noodles  and  dumplings. And  these  aren't  just  taking  these  to  show you  investors  and  taking  them to  food  shows  to  get  people  interested, But  it's  not  just  for  demonstration. You  actually  have  products on  the  market  today. Hardy  is  our  brand  name for  the  biomass  that  we  sell. You  can  scan  the  Qr  code  or  go  to  Be Foods.com  That's  our  food  company. But  you  can  see  these  products. We  don't  have  them  in  storage  yet, but  they  are  available  online. So  you  can  just  buy  the plain  algae  powder  if  you  want to  put  that  into  your  own  cooking  at  home, we  have  pasta,  green  pasta. And  then  our  newest  thing  is  these, these  all  pork  dumplings. Like  I  said,  I'm  not  a  vegetarian, but  they  are  really  good. We've  taken  them  and  given  out hundreds  of  them  at  different  shows. Unfortunately,  you  couldn't  bring  them  today, but  you  guys  combine  them, but  they  are  really  good. Everybody's  really  great  feedback on  them  and  people are  really  excited  about  them. That  pretty  much  give you  an  overview  of  who  we  are. If  you  want  to  go  to  our  website, you  can  look  up  more  about  the  company. We  also  have  some  links  that  we  recently featured  on  ACNN  show about  the  future  of  food. One  of  our  founders,  Miller  Tran, talked  about  how  we  fit into  the  food  ecosystem. Last  two  things  is  our  company came  from  academia. A  lot  of  us  spent  a  lot  of  time  in  academia. We've  done  collaborations  of  academia  before. If  you  have  any  ideas that  you  think  can  work  at scale  or  any  strains that  you  think  can  work  at  scale, we'd  love  to  hear  about  it  and talk  about  how  we  could  cloud  rate. The  other  thing  is we're  hopefully  expanding  soon, so  that  means  we'll  probably be  hiring  tech  early, but  also  maybe  some  scientists. So  if  you're  looking  to  get  into this  industry  and  you  want  to  stay  with clammy,  come  talk  to  us. We  are  in  San  Diego. I  know  that's  a  rough  thing. It's  sunny  all  the  time,  but if  I  can  survive  there, anybody  probably  can,  so  it's  not  too  bad. I'll  take  any  questions for  being  ahead  of  time. Time. Thanks. Totally  avoid  strain. That's  what  we  started  off  with. Like  you  said, it  was  all  the  recount  thing, you  know,  with  the  food  thing. And  when  we  transitioned  into  that,  you  know, we  had  to  do  the regulatory  in  the  United  States,  at  least, I'm  sure  everywhere  pretty complicated  and  the  rules are  not  very  well  established. So  that  would  just  be  another  headache. We  probably  will  get  into  that  again  soon. Probably  when  if  we  are  expanding  soon. So  Yeah,  you  know,  not  right  now. Enter  the  top  mentioned  you  grow  your  algae in  dark  fermentation  and one  reason  is  to  keep  the  south  arrow. How  would  keep  the  south  arrow and  also  Cln  grow  in? By  doing  that  reduce the  cost  by  making  carbon. It's  not  the  darkest, you  haven't  enclosed  fermenters. That's  what  keeps  the  sterile. And  do  all  this  extra  stuff  to keep  the  fermentor  sterile  light. When  you  go  really  high  densities, just  stick  a  light  in  there. That  light  so  far  once  you  get  to a  certain  density  I worked  at  another  algae before  you're  going  in  and  ponds. And  those  ponds  don't  exist  anymore  with that  company  because  it  just doesn't  make  sense. I'm  sure  somebody  eventually will  make  it  work, but  the  landscape  is littered  with  algae  companies that  tried  to  make  that  work. We're  skipping  that  and moving  ahead  to  getting  products. And  as  technology  gets  out, I  guess  we'll  look  at  everything. But  right  now  we're  doing everything  with  fermentation. Just  taste  different. I  don't  know,  I really  haven't  tasted  the  powder. I've  always,  when  they've  asked, I've  always  said,  I  don't think  my  palate  is  delicate  enough. I've  tasted  the  foods  that  they  make. And  usually,  usually  it's in  the  middle  of the  afternoon  when  you  need  a  snack. Anyway  that  the  food  scientists have  something  for  us  to  test. As  far  as  Takayasu, it  tastes  pretty  close  to get  phototrophic  growth.  I  don't  know. We  used  to  have  caros  spin  down the  whole  part  of  ways  you get  so  little  biomass. So  it'd  be  really  hard  to get  enough  to  compare,  I  guess. Thank  you.  Question  here. From  microbiology  engineering  background, when  we  have  this  fermentation  engineering on  one  of  the  biggest  costs associated  with  downstream Pic  and  processing, that's  actually  where  your  money  spent on  that. Back  to  you  consider  put  clay on  demobilized  printed  structure. Then  you  can  actually  have  a  mixture  of you  can  directly  make  this  alba  pasture. So  Pompe  question,  I  mean, I'm  not  I'm  not  the  engineer, I'm  just  a  molecular  biologist. So  maybe  one  of  our  engineers could  talk  about  that a  little  bit  more.  I  don't  know. I  mean,  I  think  we've  got  it  to a  scale  that  we  can  grow it  improvements  beyond  that. I  guess  that's  not,  that's  beyond  my  paper maybe  assumption  we  all have  is  that  when  we  grow climbing,  we  don't  reach  high. We  initi,  high  density,  we  have  to  life. And  so  how  do  you  deal  with  that? What  densities  do  you  reach? So,  like  I  said,  100, 150  g  per  liter, definitely  over  100  is  pretty  easy, The  fed  and  everything. That's  not  my  permal  per  how  many? Probably  I  would  assume that'd  be  like  10101011. Why  is  it  2  g  per  liter? I  think  that  that's  about  ten  of  the  eight, so  I'm  guessing  that  would be  100  more  than  that. We  don't  usually  bother  counting  cells. It's  pretty,  it's  hard  enough  to  get  Libor or  Lvds  total  match. But  your  high  her  250  G, so  often  what  happens  when  you by,  a  lot  of  it  is  oil. I  imagine  you  can  only  get  up to  those  densities  if there's  a  substantial  part  of that  way  you're  legally  separate. I  don't  know,  I  wish  we  had  higher  oil. That  would  probably  be  good  actually. But,  you  know,  when  we  test the  biomass,  usually  not. That  was  part  of  the  physics. Question.  Thank  you.  After  you  don't  use, that's  why  you  got  stem. Like  I  said,  I  think  there's  a  lot  of benefits  and  there's  things that  you  just  can't  do  in  East. And  if  you  want  something  green, that's  pretty  hard  to  do  in  yeast,  I  guess. So  There's  a  lot  of  other  things, we're  just  scratching  the  surface  also, you  know  the  clinical  data  as has  been  used  as  an  ingredient  forever. I  don't  think  they  have the  data  that  we  have  on  that. I  know  it's  in  so  many  were Nessa  also  maybe  pull scale  but  if  you  have  that  as a  major  people  are  going  to, you  say  all  people  are  like, thank my  question to  talk  about  transpose. Mobile  can  call,  go,  move. And  so  I'm  going  to  go  through  three  parts. One  is  rests. I'm  going  to  introduce  the  transposable library. Then  you're  going  to  touch  on two  you  can  do  with  that. One  is  to  look  the  economic  distribution  of revise  centers  and  the  other  is  to talk  about  active  elements are  I'm  not  really going  to  introduce  transpose. You're  on  board  with  the  fact  that they're  interesting  and  important. I'm  going  to  start  by  introducing a  really  quick  history  of transposable  discovery. The  first  elements  we'll discover  is  actually  actively transposing  elements  jumping  into genes  in  lab  strains  including lever  ten  years  ago  now. And  that's  really  the  iceberg  transposons, they're  in  the  genome. And  once  we  have  a  genome  sequence, it's  possible  to  mine  that and  find  multi  popular  pets. Annotates,  because  clam  is  bio, genetically  sophistic. Any  plants  that  most  people were  studying  at  the  time, actually  a  lot  of  those  transposons turned  out  to  be  completely  new. So  a  lot  of  new  transposon  biology come  out  of  the  clamped, this  accommodated  in  direct  base  libraries. If  you've  ever  set beta  transposons  and  clams, it's  probably  where  you've  got  them. This  is  119  transposon  families that  have  been  manually  generated. This  was  all  done  in  the  version  three genome  some  time  ago and  we  weren't  really  sure how  complete  this  library was  and  I  set  out  a  ph  data, try  and  answer  that  question, this  one  isn't  transposable  library. Essentially,  we  try  and  capture  all  of the  repeats  that  are  in  the  genome. And  the  key  thing  is we  make  consensus  sequence. You  have  a  repeat  in  molted  multiple  copies. You  can  combine  the  molts  into one  representative  sequence  based on  how  that  sequence  books, transposable  elements  are  incredibly  diverse. There  are  lots  of  different  mechanistic  ways to  move  yourself  around  the  genome. We  look  at  that  transposed can  actually  classify  it. This  certainly  does  require managerationcause. Most  times  when  you  do  this  in  a  plant, someone  has  done  that  somehow organism  and  you  can  take that  information  to  transfer the  plant  bonuses  so  far  away. Really  if  you  have  to  look  at them  as  say  many  of these  transposons  end  be completely  new  scientists, at  least  Just  to  give  you  an  example, Tcr  one  was  discussed 25  or  more  years  ago  as  an  act  in  the  lab, is  present  in  four  copies. And  just  show  you  the  terminal ends  of  these  copies. These  are  different  positions. The  planing  sequence,  you  can  see  the  Ali, these  four  copies  are  completely  identical. We  can  make  a  consensus  sequence to  capture  that  very  easily. There's  AG  in  the  middle  which  show  you, and  it's  ADNA  transposon  as these  voted  for  Peg, GCTCC  and  so  on. And  you  can  also  see  that  many  transposons opposed  duplications, there's  actually  baseplication  on each  side  of  these  elements. You  can  take  all  of  that  information. Tcs,  ADNA  transposon, But  no  unusual  type  of  DNA  transposon. Now  we  can  look  at  it,  it's  actually  part  of this  unusual  group  that was  only  described  a  few  years  ago. I  did  this  multiple  element  for group  genome  took  about  four  months times  the  Hd  do  that. And  this  is  the  default  is  what  we  rely  on, hasn't  really  been  touched since  late  2000,  2000, 8,009  And  the  updated  library that  I've  produced  here, the  transposon  biology  group, Transposons  hierarchically  families  with the  actual  transposing  elements themselves  and  group. An  example  is  the  family  is a  superfamily  and  DNA  transposed. This  really  expanded  things. We've  got  more  than  twice  as  many  families. Lots  of  more  diversity. And  the  reason  I never  published  this  libraries because  actually  some  of  these  you  again, so  I'm  busy  describing  those  in the  Nolan  plans  and all  sorts  of  things  that  narrowly  found, in  terms  of  the  plan, you  add  up  all  the  consensus  sequences. But  if  you  actually  look  at the  total  percent  of the  genome  that  is  covered, only  goes  up  at  50%  total  megabases, over  10%  The  red  based  library  has a  lot  of  the  really  high  number  transpose, but  there's  a  lot  of  transposes  that  are low  numbers,  that  aren't  in  that. You  want  to  say,  I  haven't  got the  figure  here  for  it, but  it's  not  just  basis in  the  genome.  It's  also  genes. 1,000  Genes  in  the  genome are  encoded  by  transpose  elements, many  of  them  are  expressed  in version  five  of  those  were  just  in  the  next, you  couldn't  really  distinguish  them. In  version  six,  we  went through  quite  a  lot  of  Tas, Tacos,  transposable  element  genes. So  you  can  separate  the  need to  one  way  of  visualizing  that  pig. This  is  called  a transposable  element  landscape. You  might  have  seen  this  pagare for  human  genome  before, and  basically  the  consensus  sequence is  capturing  all  of  that  information. For  one  transpose  transpose  element died  5  million  years  ago. Every  copy  of  that  was  degraded, randilynplot. The  distance  divergence  from the  consensus  sequence  older  the  transposon, the  more  divergence  still in  the  human  genome. This  is  a  shape  because  we  have  a  lot of  transposons  in  the  Cmo, there's  pretty  much  nothing  there. If  you  see  a  transposon, it's  probably  recently active  or  still  actually active  in  the  species, and  I'm  not  going  to  show  it, But  when  you  look  at polymorphisms  amongst  field  strains, we  can  see  that most  transposons  in  the  reference  genome  are actually  polymorphic  amongst  field  strains. The  other  really  amazing  thing here  is  diversity, Pluto  diversity  of  transposons, transposons  and  blue  lines  Ts  familiar  with? There  are  lots  of  exotic transposons  and  so  yeah, based  is  incomplete  and so  we  need  transposons  you  can  get from  this  is  a  supplementary  file in  version  six  genome  paper. I'll  hopefully  publish  it  one way  and  coming  T's  are incredibly  heads  and almost  all  of  these  T's  probably  exhibit evidence  of  recent  activity, and  that's  probably  because Olds  just  last  in  the  genome. They  just  get  pushed  by  selection. Very.  Okay,  So  I just  have  one  slide  on  section two  and  I've  lost  the  title. That's  the  pa,  specular  hopefully  makes  up. And  so  this  is  just  a  subset  of prosogenomic  distribution  of  repeats, repeats  gray, and  they  all  start  on  top  of  each  other. Most  S  are  in  blue. Then  what  I  want  to  point  out here  is  the  green  element  here. Generally,  there  are  contents not  that  high  throughout  the  chromosome, but  there  are  particular  reaches  of the  chromosome  and  usually  one  cluster  is very  obvious  chromosome  and it  turns  out  it's  probably  centre. This  had  been  matched  slightly before  through  and  the  notice  that there  were  these  reverses  s when  we  put  this  library  on  top  of  it  became very  obvious  that  it's just  one  particular  family that  is  completely  enriched and  it's  not  found  anywhere  else. And  there  was  a  poster  yesterday which  did  find  sentarme, which  is  very  nice. Point  out  that  these  accolate along  with  subdues, that's  for  black  squiggles. To  summarize  that,  a  very  quick  part,  two, I  didn't  show  it  there  but transposons  finds  genic  reaches most  trans  genome  but actually  transect  transposons  centres, a  limited  part  of  genome  but  colder the  transposon  is  found  in  there. In  particular  it's  this  one  line and  what  I  didn't  mention  there  is this  element  is  actually  homologous  to centric  alba  is  800  million  years  away. So  this  underground  is something  interesting,  involves  cents. That's  an  interesting  question that  we  don't  know. Okay,  so  final  couple  of  minutes, active  transposes  in  the  last I  really  over  simplifying  phase. But  what  we  can  do  now, we  have  a  lot  of  genome  that  many  of  the  sec, genome  specific  laboratory  strains. We  also  have  a  lot  of  luminate  data. I'm  sure  you're  aware the  laboratory  strains  are interrelated  then  in  the  last  75  years. But  we  can  now  call mutations  by  comparing  the  genomes, comparing  the  illuminate  data, and  we  can  find  the  right  variants that  we  can  call  the  mutations. And  if  the  transposes call  those  active  elements. This  is  all  well  and  good. However,  We  have  no  idea what  the  generation  number  is, We  have  no  idea  how  people  put  these. It's  interesting  that  people  in  the  group this  room  transposes  on  that. Please,  with  that. So  we  did  an  experiment  at  the  end  of my  Phd  where  we  grew to  field  estimates  for  100  generations. And  then  we  resequence line  of  bio  and  that  gives  us an  altimeters  transpose  onto  moving  around. Okay. So,  I'm  going  to  just  briefly  go  through some  results  from  the  Tory  strains. And  so  we  can  find  those  early  observations that  describe  transposes. We're  up  to  60  transposons  that  we've seen  in  the  last  strains. And  that  list  you  can  find  in  source  most  of these  families  we  found only  actually  inserting  once  or twice  in  one  or two  strains  that  we  looked  at. So  they're  active  but they're  probably  not  transposable  that  much. Others  like  RC  one, we  found  inserting  plenty of  times  in  most  strains. So  there  are  some  that  are  actually more  consistently  active. What  I  think  is  more  interesting  is occasionally  in  this  one  particular  example, we  found  one  active  transpose  CLC that  had  never  been described  as  active  before. In  some  strains  it's  just  explicitly  active, but  in  those  strains  silent. We  found  this  in  CC  4532  genome  sequence and  it  caused  more  than  80  insertions. And  that  in  more  than  a  negabasicseequence is  quite  a  big  element  as  16  Vsat  to accumulate  shot  is  really interesting  analysis  where  it  looks in  other  strains  with  illuminate  data  to try  and  find  this transposable  element  jumping. We've  found  it  in  CC  4532, but  it  actually  turns  out  there  are  lots  of insertions,  some  of  which  shared. And  you  can  use that  information  complex  to  look  at, but  you  can  use  that  information  and  you can  apply  it  onto  a  strain  history, can  see  that  as  strains  that  we  moved  around. When  you  see  this  G  seven  A, this  is  a  case  where  we  actually found  that  this  transpose become  active  and  then  we can  count  up  a  number  of  decisions. It's  become  active  several  different  times. Cc  one  to  583  of  these  decisions. When  she  does  our  CC one  to  five  you  might  have  ACC 15  that  was  never  activated  yet. This  is  happened  lab  just  very,  very  quickly. We  also  look  to  the  field  strains. Experiment  graphic  lines,  a couple  of  very  points, all  different  transposons. Again,  we  have  some  that  are  active  low  rates and  some  that  are  really, really  highly  active. Interestingly,  the  other  one, we  already  have  four  replicates, but  there's only  one  active  transport  in  this  line. There's  a  huge  amount  of variation  also  in  this  line. These  two  elements  cause a  lot  of  rearrangements. So  I'm  not  going  to  go  to  the  mechanism  here, but  it's  lucky  that  this isn't  a  labs  strain  because these  Gomes  straggle  just a  few  under  generations. Yeah,  just  to  summarize again  the  real  key  thing  here, all  Btr  carry  some  Ud  insertions  and  yeah, there's  a  lot  of  variability  substrates have  some  active  ones  and labs  strains  are  completely at  the  set  fields. Thank  you  very  much. We  ask  when  one  of  those, early  on  CR  one  was  isolated, came  from  a  chlorate  screen  where  cells were  killed  to  get  mutes  and nitrated  chlorate. And  44  we  looked at  and  40  of  them  were  caused  trips. So  do  you  see  a  correlation  between stress  like  and  chlorate  and  movement? I  haven't  looked  but  I  would  love  to. Yeah,  I  think  there  is  so  much  greater  data. Yeah,  city  active  but  a  lot  more  expressed. I  think  you  want  all  these  things  to  do. T  stress  are  cases looked  differences  in  expression. But  yeah,  so  you  show first  pens  which  is  to  apply  stresses. You  see  what  happens. And  I  think,  yeah,  absolutely  and  that's the  last  stresses  that, that's  maybe  why  sometimes  activate. But  yeah,  in  the  experiment  that  was also  complete  fairly  nice conditions  to  get  a  baseline. But  it's  definitely  what  we  would  expect. Results  from  models show  that  it  is  stressful. Said  transposons, paratactic speak  in  the  moment. Okay,  Can  applicants  pathogen, if  you  actually  put  them  to  call, they  tend  to  actually  be  significantly. So  my  question  is,  do  you, do  you  think  that  bet as  thousand  thousand  conditions  is  quick? Move  back  to  those  questions  like the  stress  conditions  did  you  treat, what's  the  ideas  think? I  don't  to  be  honest. Yeah,  I  think  that  would  have  come  out of  the  experimental  work and  I  just  don't  think, you  know,  I  mean,  we  don't  see  rearranges. The  last  rains  part  less  than  unusual. Particularly  restrain  even  the  ancestors rearrange  which  might  have happened  and  allow  might  have  happened. But  that's  particularly, you  see  the  insertions. The  combination  graphs. So  yeah,  we're  lucky  that's GI  want  to  concur  with  P. At  one  point  we  were  doing  screens, Muts  that  were  resistant  to metalness  to  get  things  that trip  biosynthesis  and  we  put  them  in 38  degrees  and  everything that  we  thought  actually  had  an  insertion  in. The  other  thing  that  might  be interesting  is  CT  125  has blocks  lysis  and  it  could  be  related  also to  all  of  the  insertions that  you're  seeing  in  that  strain. Yeah,  No,  I  think  that's  Yeah,  right  grounds. But  there's  so  many  motivations that  I  think  we  should  ask. I  didn't  have  a  question Requested  University  of  Maryland  Public  Work. We  did  a  transcript  analysis  a  few  years  ago. Several  eight  different  cell  conditions with  related  to  sexual  reproduction. And  we  found  several  transcripts, I  can't  remember  which  ones  they  were, that  were  dramatically  up regulated  when  games are  mixed  together  and undergo  psychic  amp  increase. We  just  add  Epic  MP. And  I  forgot,  I just  hadn't  thought  about that  data  very  much, but  it  seemed  that  there  were  a  lot  of  trans, several  transcripts  that  were  dramatically upregulated  during  the  A  process. And  then  a  little  bit  of  reading  on  that. I  found  that  that's  true in  other  organisms  too. Do  you  have  any  comments, thoughts  about  that? Yeah,  yeah, it  to  jump  from verification  because  that's  how they  make  themselves, because  they  jump  from  somewhere  else. Miles.  Yeah,  I  need  to  think  about  it. Video,  It  could  be  a  foot  out  mode, but  this  is  before  miles. This  is  well  before  that. Okay. Mas  salvation  system. But  actually  not  even  that. It's  after  they've  been  nitrogen  star then  they're  activated  P. So  it's  a  particular circumstance  during  the  lifetime. Okay.  Another  excites  but  so  Utica, he  accepted  to  give a  15  min  token  fitness  right  now. So  I  ask  you  to  present  the  library because  40  very  important. I  think  you'll  realize the  lungs  and,  and  the  multi. Okay.  Can  everybody  hear  me? Yes.  Right? So  my  goal,  my  goal  is  to just  give  you  a  very  brief  update of  the  Tut  library. Many  of  you  are  familiar  with  this  library. This  is  a  picture  of  some  of  the mutants  in  this  library. In  each  of  these  colonies, a  different  gene  is  disrupted  and the  identity  of  the  gene  is  known. The  mutants  are  distributed by  the  resource  center and  they  can  be  searched  on  a  website. I  wanted  to  first really  point  out  that the  clip  library  is the  fruit  of  a  team  effort. Because  of  the  time,  I  won't  have  time  to go  through  all  the  names, but  really  it's  been  led by  a  number  of  postdocs  in  the  lab and  outstanding  from  Veronica  Pta. The  project  was  originally  started  by Roja  and  then  led  by  Hables, currently  led  by  Alice  Vardon, the  current  team  on  the  project. We  have  two  technicians,  Pacini, Julia  Zubac  and  Michelle  Ward. Williams  is  our  lab  manager who's  also  contributing  heavily. And  it's  been  a  close  collaboration with  Arthur  Grossman, who  is  our  neighbor  at Carnegie  where  we  started  the  project. As  well  as  the  Mi  Resource  Center  which has  pele  and  serling  silo  and clad  and  a  number of  very  dedicated  technicians and  made  the  project  possible  over  the  years. I  want  to  briefly  say  that  we're  very grateful  to  the  community for  your  interest  in  the  project, in  your  support  of  the  project, and  your  use  of  these  mutants. I  didn't  have  time  to  update  this  slide, but  there's  been  a  steady  demand over  the  years  for  mutants. And  from  what  I  understand  from  Pete, there's  over  6,500  mutants  that  have  been distributed  to  over  200  laboratories  so  far. I'm  going  to  skip  some  slides  to  stick  to five  minute  limit  here.  Right? Okay. So  the  resource  has  been  great,  but We've  been  very  aware of  a  number  of  limitations. And  one  of  the  limitations  has  been  that that  there  is  not sufficient  coverage  of  many  genes. And  in  many  cases, there's  only  one  allele available  for  many  genes. Which  makes  it  challenging if  you  want  to  really  establish confidently  a  relationship  between a  gene  and  the  associated  phenotype. So  we  are  in the  progress  process  and thanks  to  the  support  of  the  community, we  are  in  the  process of  expanding  the  Cent  Library. So  this  is  kind  a  representation of  the  existing  library  in  terms  of the  percentage  of all  genes  genome  that  are  covered with  95%  confidence  insurgents  in  five  Trs, introns  and  exons,  which  are the  feature  types  that  ten disrupt  gene  function. And  you  can  see  that  there's only  about  90%  of  genes  that  are represented  by  three  or  more  alleles. Which  in  our  experience  is  what  you  need  to establish  a  high confidence  genotype  relationship. And  fewer  than  50%  are  represented by  just  one  or  more  high  confidence. Our  goal  is  to  just  expand  this possible  to  make  the  resource more  valuable  to  the  community. And  we  give  you, I  want  to  very  briefly  introduce  what  we're doing  in  this  particular  expansion. One  thing  that  you  should be  aware  of  it  that  the  new  mutants  are being  generated  in a  different  background  strain and  with  a  different  selection  marker. The  idea  is  to  make the  resource  valuable  in ways  that  it  wasn't  before. And  also  to  the  new  mutants  capable  of crossing  with  the  previously existing  mutant  very  easily. This  is  a  little  bit  about the  new  background  strain. With  advice  from  Pete, we  have  selected  the Whitman  and  G  one  background  strain. This  is  also  being  sensitive  to  the  flagella Celia  basal  bodies  side of  the  community  pipe. Plus  it  crosses  well  with  mutants from  the  existing  library  which  are  mating. Pipe  minus  it  is  one.  It  is  age. It  has  strong  negative phototaxis  as  vigorous  swimming. Normal  ultrastructure as  far  as  we  can  tell  has phototrophic  growth  that's  comparable to  our  existing  strain. And  I  won't  go  into  all  the  details, but  we're  in  the  process  of making  mutants  in  the  library. In  this  background  strain we  have  a  collection so  far  of  about  222,000  colonies, and  we're  pretty  far  along  the  way to  actually  having  all  the  insertions. We've  collected  all  the  high  throughput  data and  we're  in  the  process  of  analyzing  it. It's  our  expectation  that we  hope  to  deliver  to  the  community. We  hope  that  you  will  be  able  to order  strains  from  this  resource. And  within  the  next  year, let's  see,  14,  15. And  you  should  be  able  to, you're  all  warmly  invited  to  come  and visit  the  Labra,  see  the  resource. Right. And  so,  yeah, we  hope  to  have  it available  in  the  next  year. And  then  additionally,  we'll  be phenotyping  the  new  mutants in  the  presence  of  small  molecules. So  if  you  have  a particular  small  molecule that  you're  interested in  and  you  would  be  interested  in knowing  which  mutants  or  which  genes, when  mutated  confer  sensitivity or  enhanced  growth  in  the presence  of  the  small  molecule. Please  let  us  know. We'd  be  very  happy  to  collaborate  with  you. Finally,  here's  our  summary  slide. Mutants  can  be  searched  on  the Camut.org  and  Phase  websites. Many  mutant  phenotypes  are  already available  under  many  growth  conditions. Also  on  the  Clmulatarytorgbsite, we're  expanding  the  library  using a  new  strain  and  much  better  mapping, which  we  hope  will greatly  increase  the  coverage of  high  confidence  destruction  alleles. And  we'll  be phenotyping  mutants  in  the  next  year. We're  happy  to  collaborate on  the  chemical  of  interest. That's  it. Monition  I  think. Yes,  we  question  over  there  on the  way  happen  after  that, is  that  I  mean  were  always. But  I  would  stay  here  and a  couple  of  people  stay  here to  discuss  the  community  resources. We  thank  you  so  much  for  you mentioned  you  mentioned  more  matter. Yes,  absolutely.  Right. So  this  is  going  to get  a  little  bit  technical, but  basically  the  insurgions, clammy  nutrients  are  quite  messy. Right? And  so  these  either  cassettes  that  have unique  DNA  barcodes  in  them  and  very often  what  we  observe  is  one  end  of the  cassette  is  truncated. And  then  additionally,  another  challenge that  we  have  is  that  we see  next  to  our  insurgents, we  have  jumped  DNA  which  is  genomic  DNA. What  we  think  it's  genomic  DNA from  liced  cells  that got  electrocard  into  the  cell along  with  the  cassette. And  so  if  we  sequence  out  of  the  cassette, don't  sequence  far  enough, we're  sequencing  climate,  genomic  sequence. And  we  might  be  mistaken  to  think  that the  whole  cassette  is inside  this  orange  region  of  the  genome. But  in  fact,  the  entire  thing is  in  the  blue  region  of  the  genome. This  has  been  a  major  challenge  for  us. One  of  the  reasons  why  the  current  library, we  don't  have  very  high  confidence  in  many  of the  Ut  incursion  mapping  site. The  previous  mapping  approach was  done  by  using an  aluminum  paired end  sequencing  based  approach. We  amplified subsequence  between  the  cassette, including  this  unique  barcode out  as  far  as  we  could  go. And  then  we  did  paradox  sequencing  to  get the  barcode  sequence  and the  Frank  genomic  sequence. But  this  was  limited  because  we can  only  go  up  to  our  average about  500  bases  and the  most  extreme  case  about one  K.  But  still there  were  some  jump  fragments that  were  longer  than  that. Sometimes  you  get  several  jump  fragments  and sequence  The  new  approach. This  is  been  pioneered  by Alexa  leading  the  project. We're  now  able  to generate  much  longer  fragments. The  medium  size  is  2.5  KB, but  they  rather  there  are  six  KB and  we  sequence  that  by  pack  bio. This  is  very  powerful because  not  only  are  we  going  farther, but  we  actually  know the  full  order  in  which  the  sequences  are. So  if  you  have  multiple  junk  fragments, we  know  what the  sequences  that's  furthest  away  from the  cassette  and  is  therefore  most likely  to  be  the  insertion  site. And  additionally,  we're  able  to  sequence backwards  in  the opposite  direction  of  the  cassette. And  then  we're  able  to  compare the  genomic  sequences  that  we get  on  both  sides  to provide  very  high  confidence that  we  have  the  insertion  site accurately  as  you  mentioned. So  I  think  we  can  start  the  discussion  now. I  have  few  slides  were. Yeah,  let's  give.