3
3
3
3
3
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is  a  model  organism  for  Archaea,  which  affords  researchers  the  beneficial  quali$es  such  as  (1)  producing  methane  used  as  biogas  and  (2)  manufacturing   isoprenoids  as  precursors  for  high-­‐value  biochemicals.  However,  there  are  few  gene$c  tools  available  to  metabolically  engineer  Archaea.  Our  goal  is  to  develop  some  useful  tools  for   synthe$c  biology  of  Archaea.  Building  on  our  past
projects,  which  created  and  characterized  an  mCherry  reporter  system  and  a  recombinant  mutant  making  geraniol,   our  team  is  now  working  to  (1)  create,  characterize  and  model  a  ribosome-­‐binding  site  (RBS)  library  using  the  mCherry  reporter  system  and  (2)  model  geraniol  produc$on  of  the   recombinant
using  flux  balance  analyses.  Addi$onally,  our  team  has  ini$ated  an  Archaeal  InterLab  Study  to  further  characterize  the  reproducibility  of  our  mCherry   reporter  system.
Archaeal   Ribosome   Binding   Site   (RBS)   Library
Development:
2
Inspired  by  the  iGEM  HQ
InterLab  Measurement  Study,   we  extended  this  type  study  into  the  realm  of  Archaea.  We   used   mCherry   extracts   from   our   developed
transformants  (
).  Our  goal  was  to  determine  if  our   mCherry   quan$fica$on   protocol   was   reproducible   among   many  different  labs.  Collected  data  from  par$cipa$ng  teams
(
and  data  analysis
are  shown  in
.
Using   the   metabolic   model   for
that   our
2014   team   amended   by   adding   geraniol   metabolites   and   reac$ons,   we   discovered   a   significance   in   the   CO
(
).
2
/NH
4 ra$o   for   the   produc$on   of   geraniol     via
Figure  6 .  Flowchart   depic$ng  specific   constraints  tested,  as  well   as  their  flux  balance   results
A   con$nuing   project   for   our   iGEM   Team,   developing   a   library  of  varying  transla$onal  efficiency  for  Archaea,  is  a   tool   missing   in   Synthe$c   Biology.     Previously   we   characterized   a   na$ve   and   two   theore$cal   sequences
(
-­‐2).   This   year   we   created   a   new   part:
(
shows   the   representa$ve   mutants   from   our   library   that   have   met   standards  of  cul$va$on,  screening,  and  sequencing.
(
Figure  1.   The  steps  through  which  our  transla$onal    library  development  occurs.  From  L  to  R:  anaerobic   transforma$on,  PCR  and  sequencing  of  mutant,  mCherry  extrac$on  and  matura$on,    and  collec$on  of   fluorescent  data.
Random   oligonucleo$des   were   cloned   into   the   RBS   upstream   of   the   gene   encoding   mCherry   in   a   methanocccal   expression   vector.   Clones   were   picked   and   the   oligonucleo$de   iden$fied   by   PCR   and   sequencing.   Fluorescence   was   then   determined   to   evaluate  mCherry  expression  (
).  In  addi$on,  the   measurement   reproducibility   was   determined   through   the  Archaeal  InterLab  Study.
P hmvA
RBS mCherry  Reporter
C  A  G   T  T  A G  C  G  C  T   A    T    G
.   Transcrip$on   unit   including   promoter   hmvA
),   ribosome   binding   site   (RBS),   and   mCherry   reporter  gene  for  Bba_K1635000.  The  fourth  base  in   the  RBS  (designated  in  red)  has  a  G  to  T  transversion.
180
160
A
Figure  4.  (L)   Process  overview  for  conduc$ng  our  Archaeal  InterLab  Study.   (R)   List  of  teams  that  par$cipated  in  our
Archaeal  InterLab  Study
UGA
MIT
Stony  Brook
UCSF
Carnegie  Mellon
Genspace
Columbia  NYC
UGA
100%
MIT
83%
100%
Stony  Brook
90%
70%
100%
Table  1.
Correla$on  matrix  of  mCherry  fluorescence  data  across  7  par$cipa$ng  iGEM  teams  aeer  removing  individual   outlier  data  (see  below  for  the  ESD  test  table).
UGA
MIT
UCSF
Carnegie  Mellon
Genspace
Columbia  NYC
2
Stony  Brook
L1C8   L1C13 L1C15 L1C18
0.55
0.48
0.93
0.50
0.66
0.41
0.76
0.56
0.65
0.43
0.87
0.63
0.39
0.53
0.77
0.49
0.44
0.43
0.33
0.62
0.50
0.50
0.77
0.55
0.06
0.74
0.44
0.81
0.22
0.50
0.79
0.76
0.70
0.33
0.51
1.21
0.96
0.70
1.06
0.54
0.61
0.56
0.42
0.76
0.71
0.32
0.81
0.78
0.37
0.76
0.66
1.11
0.61
0.45
0.81
0.94
0.71
0.63
0.71
0.66
0.60
0.37
1.23
0.96
0.72
0.80
0.45
0.57
N.A.
1.08
1.08
0.63
0.56
0.93
0.10
0.45
0.57
0.65
0.45
0.50
0.63
0.50
0.90
0.58
Genspace
12C2
0.53
0.52
0.49
0.47
0.48
0.47
0.16
0.63
0.58
0.64
N.A.
N.A.
0.69
N.A.
N.A.
0.49
N.A.
N.A.
0.58
0.55
0.54
UCSF Carnegie  Mellon Genspace Columbia  NYC
79%
81%
69%
66%
73%
68%
82%
94%
64%
100%
62%
91%
100%
61%
70%
69%
100%
73%
76%
63%
65%
100%
L2C15 L2C16 M3C1 L2C12 15C2
0.80
0.86
0.96
0.73
0.69
0.85
0.74
1.09
0.70
0.62
0.73
0.71
0.96
0.71
0.93
0.73
0.77
0.91
0.58
0.66
0.76
0.71
1.20
0.79
0.62
0.84
1.14
0.89
0.70
0.96
0.81
0.90
0.76
0.38
0.61
0.97
0.86
1.27
0.85
0.70
0.82
1.17
0.97
0.73
0.97
0.79
0.90
0.87
0.77
0.92
0.62
0.69
1.07
0.74
0.96
0.73
0.97
1.06
0.76
0.79
0.68
1.03
1.13
1.13
0.93
0.62
0.80
0.92
0.95
1.04
0.95
1.02
0.95
0.73
0.87
0.61
0.74
0.92
0.75
0.75
0.53
0.79
1.21
0.80
0.74
0.87
0.81
0.87
0.77
1.01
0.68
1.00
1.00
0.69
0.84
0.90
0.84
1.24
0.87
0.67
0.82
1.03
0.76
0.71
1.00
Significant  outliers  (p<0.05)
Significant  outliers  (p<0.05)
Table  2.   ESD  (Extreme  Studen$zed  Deviate)  test  to  iden$fy  individual  outlier  data  obtained  for  each  mutant  colony.
The   top   row   represents   10   mutant   colonies,   while   each   column   represents   triplicate   mCherry   fluorescence   measurements  from  each  team  for  the  corresponding  colony.  Fluorescence  values  were  standardized  into  percentage   values   rela$ve   to   colony   M3C1,   by   making   the   average   fluorescence   values   for   this   colony   1.0   or   100%.   Both   significant  and  possible  outliers  were  removed  in  subsequent  analysis.  N.A.,  not  available.
140
120
100
80
60
40
G
T
T
TA
G
C
T A
C
We   used   UTR   Designer [1]   to   model   our   mutated   RBS   sequences   in   order   to   determine   if   their   prokaryo$c   transla$on   predictor   could   be   applied   to   methanogens,   specifically   our   organism
.   We   inpuoed   our   sequence  into  the  tool,  and  the  model  predicted  the  Gibbs   free   energy   (∆G
UTR
)   as   well   as   a   protein   expression   level
6
4
20
T
2 y = -0.655ln(x) + 11.243
R ² = 0.0086
0
0
.   Point   muta$on   coverage   of
transformants   that   met   cul$va$on,   screening,   and   s e q u e n c i n g   s t a n d a r d s .   T h e   s e q u e n c e
“CAGGTAGCGCTATG”   represents   the   sequence   of   the
“na$ve”  (BBa_K1383000),  with  all  other  point  mutants   standardized  to  the  na$ve’s  fluorescence  value.
-2
-4
-6 y = -1.788ln(x) + 20.501
R ² = 1
-8 Experimental Data
Theoretical Data
-10
1.E+03 1.E+05 1.E+07 1.E+09
Normalized Expression Level
Figure  5.  (L):   The  forma$on  of  the  transla$on  ini$a$on  complex  for   M.  maripaludis .  ∆G
UTR
:  the  Gibbs  free  energy   for  binding  of  the  Shine-­‐Dalgarno  (SD)  sequence  to  the  untranslated  region  (UTR)  of  the  mRNA.   (R)   Predicted  ∆G versus  normalized  expression  level  for  both  their  theore$cal  values,  and  our  experimental  values.
UTR
Limi$ng   Nitrogen:   No   change   in   geraniol   produc$on   or   specific  growth  rate  (
):
Table  3 :  Reducing  NH
4 specific  growth  rate*)
&  maintaining  CO
2
and  observing  change  in  geraniol  produc$on:  (with  same
Excess  Carbon:  Increases  geraniol  produc$on  and   specific  growth  rate  (
):
Table  4a:   Increasing  CO
2
&  maintaining  NH
4
and  observing  change  in  geraniol  produc$on  (with   corresponding  change  in  specific  growth  rate*).  By  increasing  the  amount  of  CO rate  increased,  but  not  the  geraniol  produc$on.
2
,  the  specific  growth
Table  4b :  Increasing  CO
2
+  maintaining  NH
4
and  observing  change  in  geraniol  produc$on  (with  same   specific  growth  rate*).  By  increasing  the  amount  of  CO there  was  an  increase  in  geraniol  produc$on.
2
and  maintaining  the  specific  growth  rate,
•   Crea$ng  and  selec$ng
mutants:  Rebecca  Buchanan  (RB)
•   Sequencing  analysis  of  mutants:  RB,  Lucas  Bougang  and  Anjana  Kumar
•   Fluorescence  measurement:  Hirel  Patel,  John  Buchanan  and  RB
•   UTR  modeling:  Lucas  Bougang;  Wiki:  RB  and  all  members  above
•   Archaeal  Interlab  and  outreach:  Steven  Kodish  and  Akshay  Chandora.
•   Metabolic  modeling:  RB;  Funding:  Walter  Asencios
•   Mentors:   Dr.   Zhe   Lyu   and   Narendran   Sekar   being   essen$al   to   the   quality  and  comple$on  of  our  project
•   Advisors:  Dr.  William  Whitman  advising  us  along  the  way  and  opening   up   his   lab   and   $me   to   teach   us;     Dr.   Yajun   Yan   allowing   us   to   use   fluorometer.
1
2
3
Reference:  [1]  Seo,  Sang  W.  et  al.  Predic$ve  design  of  mRNA  transla$on  ini$a$on  region  to  control  prokaryo$c   transla$on  efficiency.  (2013)   Metabolic  Engineering .   Vol.  15  pg.  67-­‐74.