Food

 Biotechnology    
FDE  403  
(5730403)  
Dr.  Yeşim  Soyer  
 Using  Reverse  Transcriptase  (RT-­‐
PCR)  in  cDNA  Cloning  
• To  clone  a  cDNA  from  just  one  mRNA  whose  
sequence  is  known,  use  type  of  PCR  called  
reverse  transcriptase  PCR  (RT-­‐PCR)  
• Difference  between  PCR  and  RT-­‐PCR  
– Start  with  an  mRNA  not  double-­‐stranded  DNA  
– Begin  by  converSng  mRNA  to  DNA  
– Next  use  forward  primer  to  convert  ssDNA  to  
dsDNA  
– Now  standard  PCR  conSnues  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 RT-­‐PCR  Can  Generate  SScky  Ends  
• With  care,  restricSon  
enzyme  sites  can  even  
be  added  to  the  cDNA  
of  interest  
• Able  to  generate  sScky  
ends  for  ligaSon  into  
vector  of  choice  
• 2  sScky  ends  permits  
direcSonal  cloning  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Real-­‐Time  PCR  

• Real-­‐Sme  PCR  quanSfies  the  amplificaSon  

of  the  DNA  as  it  occurs  
• As  DNA  strands  separate,  anneal  to  forward  
and  reverse  primers,  and  to  fluorescent-­‐
tagged  oligonucleoSde  complementary  to  
part  of  one  DNA  strand  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Fluorescent  Tags  in  Real-­‐Time  PCR  
• This  fluorescent-­‐tagged  
oligonucleoSde  serves  as  
a  reporter  probe  
– Fluorescent  tag  at  5’-­‐end  
– Fluorescence  quenching  
tag  at  3’-­‐end  
• With  PCR  rounds  the  5’  
tag  is  separated  from  the  
3’  tag  
• Fluorescence  increases  
with  incorporaSon  into  
DNA  product   Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Methods  of  Expressing  Cloned  
Genes  
Cloning  a  gene  permits    
• ProducSon  of  large  quanSSes  of  a  parScular  
DNA  sequence  for  detailed  study  
• Large  quanSSes  of  the  gene’s  product  can  also  
be  obtained  for  further  use  
– Study    
– Commerce    

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Expression  Vectors  
• Vectors  discussed  so  far  are  used  to  first  put  a  
foreign  DNA  into  a  bacterium  to  replicate  and  
screen  
• Expression  vectors  are  those  that  can  yield  
protein  products  of  the  cloned  genes  
– For  high  level  expression  of  a  cloned  gene  best  
results  oaen  with  specialized  expression  vectors  
– Bacterial  vectors  have  a  strong  promoter  and  a  
ribosome  binding  site  near  ATG  codon  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Fusion  Proteins  
• Some  cloning  vectors,  pUC  
and  pBS,  can  work  as  
expression  vectors  using  
lac  promoter  
• If  inserted  DNA  is  in  the  
same  reading  frame  as  
interrupted  gene,  a  fusion  
protein  results  
– These  have  a  parSal  β-­‐
galactosidase  sequence  at  
amino  end  
– Inserted  cDNA  protein  
Adapted  from  Molecular  Biology,  4th  
sequence  at  carboxyl  
EdiSon  by  e nd  
R.F.   Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Inducible  Expression  Vectors  
• Main  funcSon  of  expression  vector  is  to  yield  the  
product  of  a  gene  –  usually  more  is  becer  
• For  this  reason,  expression  vectors  have  very  strong  
promoters  
• Prefer  keep  a  cloned  gene  repressed  unSl  Sme  to  
express  
– Large  quanSSes  of  eukaryoSc  protein  in  bacteria  are  
usually  toxic  
– Can  accumulate  to  levels  that  interfere  with  bacterial  
growth  
– Expressed  protein  may  form  insoluble  aggregates,  
inclusion  bodies  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Controlling  the  lac  Promoter  
• lac  promoter  is  somewhat  inducible  
– Stays  off  unSl  sSmulated  
– Actually  repression  is  incomplete  or  leaky  
– Some  expression  will  sSll  occur  
• To  avoid  this  problem,  express  using  a  plasmid  
or  phagemid  carrying  its  own  lacI  repressor  
gene,  such  as  pBS  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Arabinose  Promoter  
• The  hybrid  trc  promoter  combines  strength  of  
the  trp  (tryptophan  operon)  promoter  with  
inducibility  of  lac  promoter  
• Promoter  from  ara  operon,  PBAD,  allow  fine  
control  of  transcripSon  
– Inducible  by  arabinose,  a  sugar  
– TranscripSon  rate  varies  with  arabinose  
concentraSon  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Tightly  Controlled  Promoter  
• Lambda  (λ)  phage  promoter,  PL,  is  Sghtly  
controlled  
• Expression  vectors  with  this  promoter-­‐
operator  system  are  used  in  host  cells  with  
temperature-­‐sensiSve  λ  repressor  gene  
– Repressor  funcSons  are  low  temperatures  
– Raise  temperature  to  nonpermissive  temperature,  
the  repressor  doesn’t  funcSon  and  cloned  gene  is  
expressed  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
• Expression  vectors  are  designed  to  yield  the  
protein  product  of  a  cloned  gene  
• When  a  lac  inducer  is  added,  cell  begins  to  
make  T7  polymerase  which  transcribes  the  
gene  of  interest  
• Many  molecules  of  T7  polymerase  are  made,  
so  gene  is  turned  on  to  a  very  high  level  with  
abundant  amount  of  protein  product  made  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Bacterial  Expression  System  
Shortcomings  
• There  are  problems  with  expression  of  
eukaryoSc  proteins  in  a  bacterial  system  
– Bacteria  may  recognize  the  proteins  as  foreign  and  
destroy  them  
– PoscranslaSonal  modificaSons  are  different  in  
bacteria  
– Bacterial  environment  may  not  permit  correct  
protein  folding  
• Very  high  levels  of  cloned  eukaryoSc  proteins  
can  be  expressed  in  useless,  insoluble  form  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 EukaryoSc  Expression  Systems  
• Avoid  bacterial  expression  problems  by  
expressing  the  protein  in  eukaryoSc  cell  
• IniSal  cloning  done  in  E.  coli  using  a  shucle  
vector,  able  to  replicate  in  both  bacterial  and  
eukaryoSc  cells  
• Yeast  is  suited  for  this  purpose  
– Rapid  growth  and  ease  of  culture  
– SSll  a  eukaryote  with  more  appropriate  
poscranslaSonal  modificaSon  
– Secretes  protein  in  growth  medium  so  easy  
purificaSon   Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Use  of  Baculovirus  As  Expression  
Vector  
• Viruses  in  this  class  have  a  large  circular  DNA  
genome,  130  kb  
• Major  viral  structural  protein  is  made  in  huge  
amounts  in  infected  cells  
– Promoter  for  this  protein,  polyhedrin,  is  very  
acSve  
– These  vectors  can  produce  up  to  0.5  g  of  protein  
per  liter  of  medium  
– Nonrecombinant  viral  DNA  entering  cells  cannot  
result  in  infecSous  virus  as  it  lacks  an  essenSal  
gene  supplied  bAdapted  
y  the   volecular  
from  M ector   Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
Baculovirus  Expression  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Animal  Cell  TransfecSon  
• Calcium  phosphate  
– Mix  cells  with  DNA  in  a  phosphate  buffer  
– Then  soluSon  of  calcium  salt  added  to  form  a  
precipitate  
– Cells  take  up  the  calcium  phosphate  crystals  which  
include  some  DNA  
• Liposomes  
– DNA  mixed  with  lipid  to  form  liposomes,  small  vesicles  
with  some  of  the  DNA  inside  
– DNA-­‐bearing  liposomes  fuse  with  cell  membrane  
carrying  DNA  inside  the  cell  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
• Foreign  genes  can  be  expressed  in  eukaryoSc  
cells  
• These  eukaryoSc  systems  have  advantages  over  
prokaryoSc  ones  
– Made  in  eukaryoSc  cells  tend  to  fold  properly  and  
are  then  soluble  rather  than  aggregated  into  
insoluble  inclusion  bodies  
– PoscranslaSonal  modificaSons  are  made  in  a  
eukaryoSc  manner  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Using  the  Ti  Plasmid  to  Transfer  
Genes  to  Plants  
• Genes  can  be  introduced  into  plants  with  
vectors  that  can  replicate  in  plant  cells  
• Common  bacterial  vector  promoters  and  
replicaSon  origins  are  not  recognized  by  plant  
cells  
• Plasmids  are  used  containing  T-­‐DNA  
– T-­‐DNA  is  derived  from  a  plasmid  known  as  tumor-­‐
inducing  (Ti)  
– Ti  plasmid  comes  from  bacteria  that  cause  plant  
tumors  called  crown  galls  
Adapted  from  Molecular  Biology,  4th  
EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
 Ti  Plasmid  InfecSon  
• Bacterium  infects  plant,  transfers  Ti  plasmid  to  
host  cells  
• T-­‐DNA  integrates  into  the  plant  DNA  causing  
abnormal  proliferaSon  of  plant  cells  
• T-­‐DNA  genes  direct  the  synthesis  of  unusual  
organic  acids,  opines  which  can  serve  as  an  
energy  source  to  the  infecSng  bacteria  but  are  
useless  to  the  plant  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
Ti  Plasmid  Transfers  Crown  Gall  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.    
Use  of  the  T-­‐DNA  Plasmid  

Adapted  from  Molecular  Biology,  4th  

EdiSon  by  R.F.  Weaver,  The  McGraw-­‐Hill  
Companies,  Inc.