How to bioengineer a novel system?

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• How to bioengineer a novel system?
• Obtain a sequence by PCR, then clone it into a
  suitable plasmid
• Were adding DNA, but want E. coli to make a
  protein!

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1. In bacteria transcription and translation are
   initially coupled

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• In Bacteria transcription and translation are
  initially coupled
• RNA polymerase quits if ribosomes lag too much

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• In Bacteria transcription and translation are
  initially coupled
• RNA polymerase quits if ribosomes lag too much
• Recent studies show that ribosomes continue
  translating once mRNA is complete i.e after
  transcription is done

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• Bacteria have gt 1 protein/mRNA (polycistronic)
• http//bmb-it-services.bmb.psu.edu/bryant/lab/Proj
  ect/Hydrogen/index.htmlsection1
• euk have 1 protein/mRNA

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• Bacteria have gt 1 protein/mRNA (polycistronic)
• Mutations can have polar effects mutations in
  upstream genes may affect expression of perfectly
  good downstream genes!

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Regulating transcription Telling RNA pol to copy
a DNA sequence
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Regulating transcription Telling RNA pol to copy
a DNA sequence Transcription factors bind
promoters control initiation of transcription
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Regulating transcription Telling RNA pol to copy
a DNA sequence Transcription factors bind
promoters control initiation of
transcription 1/signal gene senses
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Regulating transcription Telling RNA pol to copy
a DNA sequence Transcription factors bind
promoters control initiation of
transcription 1/signal gene senses 1 binding
site/signal gene senses
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Transcription factors Bind surface -gt base-pairs
form unique patterns in major minor grooves
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Transcription factors Bind surface -gt base-pairs
form unique patterns in major minor
grooves Scan DNA for correct pattern
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Transcription factors Bind surface -gt base-pairs
form unique patterns in major minor
grooves Scan DNA for correct pattern need 15 - 20
H-bonds 5-8 base-pairs
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Transcription Prokaryotes have one RNA
polymerase makes all RNA core polymerase
complex of 5 subunits (a1aIIbbw)
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Transcription Prokaryotes have one RNA
polymerase makes all RNA core polymerase
complex of 5 subunits (a1aIIbbw) w not
absolutely needed, but cells lacking w are very
sick
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Initiating transcription in Prokaryotes 1) Core
RNA polymerase is promiscuous
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• Initiating transcription in Prokaryotes
• Core RNA polymerase is promiscuous
• sigma factors provide specificity

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• Initiating transcription in Prokaryotes
• Core RNA polymerase is promiscuous
• sigma factors provide specificity
• Bind promoters

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• Initiating transcription in Prokaryotes
• Core RNA polymerase is promiscuous
• sigma factors provide specificity
• Bind promoters
• Different sigmas bind different promoters

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• Initiating transcription in Prokaryotes
• Core RNA polymerase is promiscuous
• sigma factors provide specificity
• Bind promoters
• 3) Once bound, RNA polymerase
• melts the DNA

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• Initiating transcription in Prokaryotes
• 3) Once bound, RNA polymerase
• melts the DNA
• 4) rNTPs bind template

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• Initiating transcription in Prokaryotes
• 3) Once bound, RNA polymerase
• melts the DNA
• 4) rNTPs bind template
• 5) RNA polymerase catalyzes phosphodiester bonds,
  melts and unwinds template

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• Initiating transcription in Prokaryotes
• 3) Once bound, RNA polymerase
• melts the DNA
• 4) rNTPs bind template
• 5) RNA polymerase catalyzes phosphodiester bonds,
  melts and unwinds template
• 6) sigma falls off after 10 bases are added

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Structure of Prokaryotic promoters Three DNA
sequences (core regions) 1) Pribnow box at -10
(10 bp 5 to transcription start) 5-TATAAT-3
determines exact start site bound by s factor
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Structure of Prokaryotic promoters Three DNA
sequences (core regions) 1) Pribnow box at -10
(10 bp 5 to transcription start) 5-TATAAT-3
determines exact start site bound by s
factor 2) -35 region 5-TTGACA-3 bound by
s factor
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Structure of Prokaryotic promoters Three DNA
sequences (core regions) 1) Pribnow box at -10
(10 bp 5 to transcription start) 5-TATAAT-3
determines exact start site bound by s
factor 2) -35 region 5-TTGACA-3 bound by
s factor 3) UP element -57 bound by a factor
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Structure of Prokaryotic promoters Three DNA
sequences (core regions) 1) Pribnow box at -10
(10 bp 5 to transcription start) 5-TATAAT-3
determines exact start site bound by s
factor 2) -35 region 5-TTGACA-3 bound by
s factor 3) UP element -57 bound by a factor
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Structure of Prokaryotic promoters Three DNA
sequences (core regions) 1) Pribnow box at -10
(10 bp 5 to transcription start) 5-TATAAT-3
determines exact start site bound by s
factor 2) -35 region 5-TTGACA-3 bound by
s factor 3) UP element -57 bound by a
factor Other sequences also often influence
transcription! Eg Trp operator
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Prok gene regulation 5 genes (trp operon) encode
trp enzymes
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Prok gene regulation Copy genes when no
trp Repressor stops operon if trp
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Prok gene regulation Repressor stops operon if
trp trp allosterically regulates repressor
can't bind operator until 2 trp bind
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lac operon Some operons use combined on off
switches E.g. E. coli lac operon Encodes
enzymes to use lactose lac Z ?-galactosidase lac
Y lactose permease lac A transacetylase
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lac operon Make these enzymes only if 1) -
glucose
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lac operon Make these enzymes only if 1) -
glucose 2) lactose
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lac operon Regulated by 2 proteins 1) CAP protein
senses glucose
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• lac operon
• Regulated by 2 proteins
• CAP protein senses glucose
• lac repressor senses lactose

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• lac operon
• Regulated by 2 proteins
• CAP protein senses glucose
• lac repressor senses lactose
• encoded by lac i gene
• Always on

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lac operon 2 proteins 2 binding sites 1) CAP
site promoter isnt active until CAP binds
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• lac operon
• 2 proteins 2 binding sites
• CAP site promoter isnt active until CAP binds
• Operator repressor blocks transcription

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lac operon Regulated by 2 proteins 1) CAP only
binds if no glucose -gt no activation
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lac operon Regulated by 2 proteins 1) CAP only
binds if no glucose -gt no activation 2)
Repressor blocks transcription if no lactose
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lac operon Regulated by 2 proteins 1) CAP only
binds if no glucose 2) Repressor blocks
transcription if no lactose 3) Result only make
enzymes for using lactose if lactose is present
and glucose is not
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(No Transcript)
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Result ?-galactosidase rapidly rises if
no glucose lactose is present W/in 10 minutes
is 6 of total protein!