The purpose of this RFC is to provide standard methodology for the measurement of the absolute strength of terminators in bacteria. Because we have characterized the performance of terminator in E. coli and used a simple equation model, it can be expressed in PoPS

Chen, Yao

He, Jiankui

Jing, Shui

Liu, Xianggeng

Qie, Boyu

Shen, Liuxing

Wu, Zishan

Zhang, Mengshi

DSpace@MIT

BBF	  RFC	  90：The	  Measurement	  of	  rho-­‐independent	  Transcription	  Terminator	  Efficiency.	  Jiankui	  He,	  Shui	  Jing,	  Mengshi	  Zhang,	  Xianggeng	  Liu,	  Yao	  Chen,	  Boyu	  Qie,	  Liuxing	  Shen	  Zishan	  Wu	  	  26	  September	  2012	  1. Purpose	  The	  purpose	  of	  this	  RFC	  is	  to	  provide	  standard	  methodology	  for	  the	  measurement	  of	  the	  absolute	  strength	  of	  terminators	  in	  bacteria.	  Because	  we	  have	  characterized	  the	  performance	  of	  terminator	  in	  E.coli	  and	  used	  a	  simple	  equation	  model,	  it	  can	  be	  expressed	  in	  PoPS.	  	  2. Relation	  to	  other	  BBF	  RFCs	  This	  maybe	  doesn’t	  relate	  to	  any	  other	  BBF	  RFC.	  	  3. Copyright	  Notice	  Copyright	  (C)	  The	  BioBrick	  foundation(2012).All	  Right	  Reserved.	   	  	  4. Standard	  terminator	  efficiency	  measurement	  kit	  for	  use	  in	  E.coli.	  The	   terminator	   efficiency	   measurement	   kit	   MUST	   include	   promoter,	   ribosome	  binding	  sites,	  reporter	  gene	  and	  vector	  backbone.	  Standard	  transcription	  terminator	  efficiency	   measurement	   kit	   MUST	   comprise	   of	   promoter	   BBa_R0040,	   ribosome	  binding	  site	  BBa_B0030,	  reporter	  gene,	  RFP	  BBa_E1010,	  GFP	  BBa_E0040.	  It	  SHOULD	  comprise	  vector	  backbone	  pSB1A3,	  a	  high	  copy	  number	  plasmid	  carrying	  ampicillin	  resistance.	   This	  high	   copy	  number	   vector	   is	   chosen	   to	   facilitate	  growth	  of	  bacteria	  step	  in	  the	  experiment	  	  5. Design	  and	  construction	  of	  characterization	  devices.	  The	  fluorescence	  produced	  by	  the	  characterization	  devices	  are	  then	  measured	  using	  flow	  cytometry	  to	  calculate	  the	  termination	  efficiency	  of	  the	  terminators.	  Experimental	  Group:	  	  (Inputs	   are	   measured	   by	   RFP	   expression	   and	   outputs	   are	   measured	   by	   GFP	  expression.)	  	  	  	  	  Control	  Group:	  	  (calibrates	  RFP	  input	  to	  GFP	  output.)	  	  6. Experimental	  procedure	  6.1 Media:	  Lysogeny	  Broth	  (LB)	  liquid	  and	  solid	  media.	  6.2 Protocol:	  a. Plasmid	  pSB1A3	  is	  chosen	  to	  be	  the	  vector	  that	  ligate	  GPF	  and	  RFP	  fragments.	  To	  protect	  the	  structural	  integrity	  of	  the	  constructed	  plasmid,	  we	  need	  to	  mutate	  a	  restriction	  enzyme	  cutting	  site	  named	  Pst	  I	  to	  Afl	  II	  using	  PCR.	  Proper	  primer	  are	  designed	  for	  this	  purpose.	  PtoA-­‐F:5'-­‐CCACCTGACGTCTAAGAAAC-­‐3'	  PtoA-­‐R:5'-­‐ATGATCATCGCCGGCGAATTCAGGC-­‐3'	  b. Check	  whether	  the	  mutation	  of	  restriction	  cutting	  site	  is	  successful.	  c. Transform	  the	  plasmid	  to	  competent	  cell	  DH5αand	  incubate	  overnight.	  d. Pick	  up	  colonies,	  plasmid	  isolation,	  and	  digest	  to	  choose	  the	  positive	  one.	  e. Amplify	   GFP	   &	   RFP.	   Do	   an	   electrophoresis	   for	   verification	   the	   PCR	   is	  successful.	  f. Use	   specific	   restriction	   enzymes	   to	   digest	   plasmid	   mutant-­‐psb1a3,GFP	   and	  RFP	  to	  get	  sticky	  ends	  and	  purify	  the	  DNA	  fragment	  after	  the	  Electrophoresis.	  Digestion	  of	  plasmid	  mutant-­‐pSB1A3	  with	  Afl	  II	  and	  Not	  I;	  Digestion	  of	  PCR	  product	  GFP	  with	  Not	  I	  and	  Spe	  I;	  Digestion	  of	  PCR	  product	  RFP	  with	  Afl	  II	  and	  Spe	  I;	  Put	  the	  tubes	  in	  37℃	   environment	  for	  4-­‐8	  hours.	  g. Ligation	  is	  needed	  to	  connect	  these	  3	  fragments	  together.	  h. Transform	  the	  plasmid	  to	  competent	  cell	  DH5αand	  incubate	  overnight.	  i. Pick	  up	  colonies,	  plasmid	  isolation,	  and	  choose	  the	  positive	  one.	  j. Use	  restriction	  enzymes	  Xba	  I	  and	  Pst	  I	  to	  digest	  the	  constructed	  vector.	  k. Ligation	  is	  needed	  to	  terminators	  to	  be	  measured	  and	  the	  vector	  together.	  l. Transform	  the	  plasmid	  to	  competent	  cell	  and	  incubate	  overnight.	  m. Test	  the	  fluorescent	  using	  the	  method	  of	  flow	  cytometry.	  7. Parts	  used	  to	  create	  the	  terminator	  characterization.	  Left Part Right Part Description BBa_E1010	   BBa_E0040	   RFP+BBa_K827086+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827087+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827088+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827089+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827090+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827091+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827092+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827093+GFP	  BBa_E1010	   BBa_E0040	   RFP+BBa_K827094+GFP	  	  8. Calculate	  terminator	  efficiency	  TE=1-­‐GE/GC	  TE:	  Terminator	  efficiency	  GE:	  Mean	  value	  of	  GFP	  from	  experimental	  group	  GC:	  Mean	  value	  of	  GFP	  from	  control	  group	  	  Under	  the	   ideal	  circumstances,	   the	  output	  of	  GFP	  and	  RFP	  should	  be	  almost	  the	  same	  and	  the	   value	   of	   TE	   should	   approach	   0.	   If	   a	   strong	   terminator	   is	   placed	   into	   characterization	  experimental	   group	   plasmid,	   the	   only	   output	   should	   be	   RFP	   and	   the	   value	   of	   TE	   should	  approach	  1.	   	  	  9. Author’s	  contact	  information	  Jiankui	  He:	   	   	   	   	   	   	   	   jiankuihe@gmail.com	  Shui	  Jing:	   	   	   	   	   	   	   	   	   betty.jingshui@gmail.com	  Mengshi	  Zhang:	   	   	   	   mengshi0928@gmail.com	  Xianggeng	  Liu:	   	   	   	   	   liuxianggeng@gmail.com	   	  Yao	  Chen:	   	   	   	   	   	   	   	   	   chen.yy@live.com Boyu	  Qie:	   	   	   	   	   	   	   	   	   boyuqie@gmail.com	  Liuxing	  Shen:	   	   	   	   	   	   shenliuxing@gmail.com	  Zishan	  Wu:	   	   	   	   	   	   	   	   zishan.woo@gmail.com  10. References [1]	  http://partsregistry.org/wiki/index.php/Part_Types:Measurement_Systems	  [2]	  Haiyao Huang “Design and Characterization of Artificial Transcriptional Terminators”. [3]	  Lars Velten, Nao”	  Units for Promoter Measurement in Mammalian Cells” 	  	  

The Measurement of rho‐independent Transcription Terminator Efficiency

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The Measurement of rho‐independent Transcription Terminator Efficiency

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