Key  Achievements  1.   Characterised   and   submi6ed   three   BioBricks;   BBa_K1027001,  BBa_K1027002  (improvement),  &  BBa_K1027003  2.   Built   models   incorporaFng   stochasFc/determinisFc   techniques   -­‐   directly  influencing  experimental  and  human  pracFces  work  3.   Compiled   detailed   impact   analysis   report   on   the   effects   of   introducing   a  syntheFc  alternaFve  to  a  naturally-­‐occurring  commodity  4.  Collaborated  with  2  UK  teams  by  producing  basic  guides  to  modelling        

E.  c(oil)i:  The  Lean,  Green  Fat-­‐Producing    Synbio  Machine  

Divita  Kulshrestha,  Elsa  Axelsdo\r,  Jessica  Birt,  Lorna  Hepworth,  Rob  Harrison,  Tim  Curd,  Ralf  Wenz,  Vun  Hiang  Tan,  Marco  Pinheiro,  Ma6  Birt  

References  Xu  et  al  (2013)  Modular  op*miza*on  of  mul*-­‐gene  pathways  for  fa8y  acids  produc*on  in  E.  coli.  Nature  Communica6ons  4:1409    Achcar  F  et  al  (2012)  Dynamic  Modelling  under  Uncertainty:  The  Case  of  Trypanosoma  brucei  Energy  Metabolism.  PLoS  Comput  Biol  8(1)  

 

Experimental    

Sponsors  

Overview  

BioBricks  

Pathway  Overview:  BioBricks  in  Purple  

CharacterisaFon    •  For  the  purposes  of  characterisa6on,  we  used  a  standard  part  from  the  registry    (BBa_K608002)  to  create  a  new  expression  construct  which  contains  a  ribosomal  binding  site  and  a  cons6tu6ve  promoter      •’Delta  9’  and  ‘Delta  12’  strains  of  E.  coli  were  fed  stearic  and  oleic  acid,  respec6vely.  Changes  in  metabolic  content  was  analyzed  using  Orbitrap  LC-­‐MS.        Results  •  Our  BioBrick  BBa_K1027002  works,  as  Delta  12  desaturase  is  conver6ng  the  substrate  oleic  acid  into  the  linoleic  acid.  This  is  an  improvement  of  a  previous  BioBrick  submiVed  by  St  Andrews  in  2012    

Our  Delta  9  and  Delta  12  BioBricks  are  •  Codon  op6mised  •En6rely  novel  gene  sequences  not  found  in  nature  •  Based  on  protein  sequences  from  Synechocys*s  sp.  PCC  603      

Samples

Iden6fica6on  of  metabolites  of  interests  

Analysis  of  desired  phospholipids    

Samples   Orbitrap  UHPLC-­‐FTMS  

Metabolic  Profile  

Metabolites  of  interest  

The  economies  of  Malaysia  and  Indonesia  rely  heavily  on  the  palm  oil   industry.  Our  project  could  have  major  detrimental  effects  on  these  countries.    However,   palm  oil   prices   are   constantly   increasing,   and   so   the  absence  of   our  project   could  have  an  even  worse  effect  on   the  global  economy.   Interna6onal  biofuel  targets  will  result  in  massive  increases  in  price  of  not  just  palm  oil,  but  all  other  vegetable  oils.    We  also  created  a  business  plan  to  outline  how  we  could  theore6cally  scale  this  project  up  to  an  industrial  scale.  We  es6mate  that  the  same  amount  of  palm  oil  could   be   produced   industrially   in   a   12.8   hectare   space   as   a   43,000   hectare  tradi6onal  oil  palm  planta6on.    

Modelling  

 

SchemaFc  Workflow:  

Uncertainty  Analysis  •  Challenge:  Lack  of  reliable  kineFc  values  for  key  reac6ons    •  Solu6on:  creaFng  model  explicitly  acknowledging  lack  of  data  and  uncertainty  •  Approach:  Monte  Carlo  sampling  from  plausible  parameter  value  distribu6ons  •  Result:  model  predicFons  with  confidence  intervals.    •  Conclusion:  unusual  and  innovaFve  modelling  strategy  for  poten6al  principled  

approach  to  handling  uncertainty  Synthe6c  Biology  always  is  at  cuang  edge  of  knowledge  and  hence  challenged  by  uncertainty.  Dealing  with  uncertainty  will  yield  model  predic6ons  to  inform  more  robust  design  strategies  for  a  wide  range  of  engineered  cellular  machines.    

Results:  The  data  contained  large  amounts  of  uncertainty;  nonetheless,  most  metabolites  where  within  a  narrow  range  –  especially  at  the  output  of  the  pathway.  This  proves  models  generated  by  this  method  are  useful  to  inform  further  systems  engineering  

Classifying  into  categories  

MulFple   simulaFons   within   100   seconds   at   ten   different  Fme   points.   Colours   visualise   concentraFons   according   to  their   amount   [mM]   (Dark   Red:   >4;   Pink:   >   2;   Light   red:>  1;  White:  1-­‐0.01;  Light  yellow:<0.01;  Dark  yellow:  <0.0001)  

Table   of   all   analysed  models   with   colour   coded   visualisaFon  according   to   qualitaFve   concentraFon   distribuFon   for   each  metabolite.  The  brighter  in  colour  a  cell  is,  the  more  olen  the  simulaFons  resulted  in  the  specified  concentraFon  interval.  

Summarized  boxplots  of  the  last  metabolite  concentraFons  (mM)  in  the  modelled  fa6y  acid  synthesis  pathway.  

Due  to  the  nature  of  our  project,  ethics  played  a  big  role.  We  compiled  a  23  page  impact  analysis  report   which   explored   the   environmental   and  economic  impacts  of  both  current  produc6on  and  our   synthe6c   alterna6ve   to   palm   oil.   We   also  discuss   ways   in   which   these   impacts   can   be  managed  effec6vely.    

Ethics  

Economics  

Human  PracFces  

Environmental  

Predicted  ex6nc6on  of  orangutans,  based  on  deforesta6on  data  analysed  on  our  Human  Prac6ces  pages  (LINK)    (2.36%  annual  loss  of  rainforest  coverage).  200  simula6ons  are  shown.  LEFT:  Predicted  6me  un6l  ex6nc6on  if  deforesta6on  carries  on  as  it  does  now.  RIGHT:  Popula6on  dynamics  if  deforesta6on  stopped  within  30  years    

Mass  deforesta6on   to  make   room   for  oil   palm  planta6ons   is   one  of   the  biggest  problems  of  our  Fmes:  •Areas  the  size  of  300  football  fields  are  being  cleared  every  hour    •Slash  and  burn  prac6ces  are  common  •CriFcally  endangered  species,  such  as  the  Sumatran  orangutan,  are  on  the  brink  of  exFncFon  due   to   this  destruc6on  of   their  habitat   -­‐  our  PopulaFon  Dynamics  models  showed  us  that  this  species  will  be  ex6nct  within  45  years   if  no  ac6on  is  taken.  

Human  PracFces  

Modelling  

Experimental  

PopulaFon  Dynamics  

Protein  Dynamics  

E.  c(oil)i  

PALM  OIL  

Aim:  To  create  a  sustainable,  syntheFc  alternaFve  to  palm  oil  by  reengineering  the  E.  coli  fa6y  acid  biosynthesis  pathway