Concepts of Molecular Biology

(120.602.01)

Lecture 10:
Reading Frame Practical

Anthony K. L. Leung
anthony.leung@jhu.edu
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Molecular Cloning
1. Prepare fragment via PCR
2. Digest vector and insert
3. Insert fragment into an expression vector
4. Transform plasmid into E. coli
5. Pick colonies with insert
6. Replicate the plasmid by growing E. coli
7. Extract plasmid for use in experiments
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Necessary components of a plasmid
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Restriction Enzymes
• Purified from bacteria
• Cleave DNA in sequence-specific manner
• Most recognize palindromic sequences
• Cuts same site on top and bottom strand
• Can leave “blunt” or “sticky” ends
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Green Fluorescent Protein (GFP)
• Isolated 1962, Nobel Prize 2008
• Used to visually track proteins and provide a Wildtype
(WT)
standard tag
• Expression
• Localization
• Pull-down
• Blotting
• &c. Mutant
• Can be added to protein of interest via
molecular cloning
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In-Class Practice: Cystic Fibrosis
Some mutations can lead to the mislocalization
or reduced expression of the CFTR protein. Wildtype
(WT)

You discover a candidate CF mutation directly

upstream of the CFTR gene.

Question: Does the mutation lead to irregular

Mutant
localization or expression of the protein?
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You have an EGFP-N1 plasmid available

HpaI HindIII EcoRI PstI BamHI AgeI

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Step 1: Synthesize insert
5
1) Design primers

GTTA
AC
2) Add restriction sites CA
TG G
C

3) Add additional nts NNN

GTT
1) For restriction enzyme AAC
NN
NN N
2) For reading frame TG G
CCA

(More on this later)

NNNGTTAACNN CCATGGNNN
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Step 2: Digest vector and insert with
restriction enzyme
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Step 3: Ligate fragments together
• Complements bind and are ligated together with T4 DNA ligase
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Step 4: Transform and plate E. coli
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Step 5: Pick bacteria colonies
• Each colony originates from a
single E. coli strain and
contains an identical plasmid
with antibiotic resistance
• Inoculate liquid culture
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Step 6: Grow bacteria (amplify plasmid)
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Step 7: Purify and transfect plasmid

Transform = into prokaryotic cells

Transfect = into eukaryotic cells
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Now let’s walk through this process:
1) Design primers

GTTA
AC
2) Add restriction sites CA
TG G
C

3) Add additional nts NNN

GTT
1) For restriction enzyme AAC
NN
NN N
2) For reading frame TG G
CCA

NNNGTTAACNN CCATGGNNN
Based on this MCS, which restriction sites added 3
to primers would ensure our protein is inserted
into the plasmid in the correct orientation?

HpaI HindIII EcoRI PstI BamHI AgeI

A. Forward primer: HindIII Reverse primer: HpaI

B. Forward primer: PstI Reverse primer: AgeI
C. Forward primer: PstI Reverse primer: HindIII
D. Forward primer: BamHI Reverse primer: PstI
Design primers to synthesize the insert 5

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG

CFTR is much larger than this, but pretend this is a whole gene…
Design primers to synthesize the insert 5
First, design forward and reverse primers for PCR (same process as lecture 3)

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
3’ GCTGTATGGGATCGTCTCTG 5’
Reverse Primer

Forward Primer
5’ AACATCACAGAAGGTCAGAGC 3’
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG

Sequence the primer will bind

1. ~20 nucleotides
2. Tm of between 55 and 65C

“Tm = 4(G+C) + 2(A+T)” or can be calculated online using a variety of algorithms

Design primers to synthesize the insert 5
Add restriction enzyme cut sites to the 5’ end of each primer

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
3’ GCTGTATGGGATCGTCTCTG 5’
Reverse Primer

Forward Primer
5’ AACATCACAGAAGGTCAGAGC 3’
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG

EcoRI Restriction site (Not complementary to the template!)

5’ GAATTC

Must be the 5’ end, adding to the 3’ end would stop PCR from working!
Design primers to synthesize the insert 5
Add extra 4 nucleotides before the restriction enzyme cut site

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
3’ GCTGTATGGGATCGTCTCTG 5’
Reverse Primer

Forward Primer
5’ AACATCACAGAAGGTCAGAGC 3’
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG

Extra bases (N=A,T,C or G)

5’ NNNN GAATTC

The extra bases are needed for the restriction enzyme to work
Design primers to synthesize the insert 5

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
3’ GCTGTATGGGATCGTCTCTG 5’
Reverse Primer
5’ A
TCG
G
AAT Forward Primer
TC
AACATCACAGAAGGTCAGAGC 3’
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG

5’
Design primers to synthesize the insert 5

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
3’ GCTGTATGGGATCGTCTCTG 5’
Reverse Primer
5’ A
TCG
G
AAT Forward Primer
TC
AACATCACAGAAGGTCAGAGC 3’
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG

You still add to the 5’ end!

GGATCC ATCG 5’

BamHI Restriction site and 4 extra bases

Design primers to synthesize the insert 5

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
3’ GCTGTATGGGATCGTCTCTG GGA
TCC
ATC
Reverse Primer G 5’
5’ A
TCG
G
AAT Forward Primer
TC
AACATCACAGAAGGTCAGAGC 3’
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG
Design primers to synthesize the insert 5

TACGGCTGAACATCACAGAAGGTCAGAGCCCGAGGGTTGAGCGGCAGGCACCCAGAGTAGTAGGTCTTTGGCATTAGGAGCTTGAGCCCGACATACCCTAGCAGAGACCTACGCAGC
3’ GGA
TCC
ATC
Reverse Primer G 5’
5’ A
TCG
G
AAT Forward Primer
TC
AACATCACAGAAGGTCAGAGC 3’
ATGCCGACTTGTAGTGTCTTCCAGTCTCGGGCTCCCAACTCGCCGTCCGTGGGTCTCATCATCCAGAAACCGTAATCCTCGAACTCGGGCTGTATGGGATCGTCTCTGGATGCGTCG

PCR

Restriction digest
Which primer pair (forward/reverse) would you use 5
to clone the follow DNA with EcoRI and BamHI
ATATTGAATGCACGTAAGAACATGCATATATA
EcoRI = GAATTC
TGCCTCCATTCACCTGTGGGACCTGCTGGAAG
BamHI = GGATCC
GGAAGGAAAGACCTGTATGTGGAACCACCTCG

A. GAATGCACGTAAGAACATGCGAATTCATCG (Tm=58C)
AGGTGGTTCCACATACAGGTGGATCCATCG (Tm=58C)

B. ATCGGAATTCGAATGCACGTAAGAACATGC (Tm=58C)
ATCGGGATCCAGGTGGTTCCACATACAGGT (Tm=58C)

C. ATCGGAATTCGAATGCACGTAAGAACATGC (Tm=58C)
AGGTGGTTCCACATACAGGTGGATCCATCG (Tm=58C)

D. GAATTCATCGGAATGCACGTAAGAACATGC (Tm=58C)
GGATCCATCGAGGTGGTTCCACATACAGGT (Tm=58C)
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Which construct will transcribe a functional
protein? (green = start codon; red = stop codon)

A B C D

GFP CFTR GFP CFTR GFP CFTR GFP CFTR

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Need start codon! No stop codon! Ok if we have redundant Need stop codon!
start codon
Design primers to clone CFTR as a GFP fusion 6

Vector Multiple Cloning Site (MCS)

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII
Design primers to clone CFTR as a GFP fusion 6

We have to remember about reading frame Designing the reverse primer

when designing the forward primer is the same as before

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Replace this sequence with the CFTR gene

Design the forward primer to clone CFTR as a GFP fusion 6

CFTR Gene ATG CAG AGG TCG CCT CTA AAA AAG GCC AGC GTT GTC TCC …

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII
Design the forward primer to clone CFTR as a GFP fusion 6
Start codon

CFTR Gene ATG CAG AGG TCG CCT CTA AAA AAG GCC AGC GTT GTC TCC …

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Forward Primer

5’ ATG CAG AGG TCG CCT CTA AA 3’ Tm = 58C

Design the forward primer to clone CFTR as a GFP fusion 6
Start codon

CFTR Gene ATG CAG AGG TCG CCT CTA AAA AAG GCC AGC GTT GTC TCC …

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Forward Primer

5’ TCCGGA ATG CAG AGG TCG CCT CTA AA 3’ Tm = 58C

Add restriction site

Design the forward primer to clone CFTR as a GFP fusion 6
Start codon

CFTR Gene ATG CAG AGG TCG CCT CTA AAA AAG GCC AGC GTT GTC TCC …

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Forward Primer

5’ ATGC TCCGGA ATG CAG AGG TCG CCT CTA AA 3’ Tm = 58C

Add extra nucleotides

Design the forward primer to clone CFTR as a GFP fusion 6

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Forward Primer

5’ ATGC TCCGGA ATG CAG AGG TCG CCT CTA AA 3’ Tm = 58C

Cloning (amplify CFTR, cut with BspEI, ligate vector and insert)

…TACAAGATCCGGAATGCAGAGGTCGCCTCTAAAAAGGCC…

CFTR start codon

Design the forward primer to clone CFTR as a GFP fusion 6

GFP codons
TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Forward Primer

5’ ATGC TCCGGA ATG CAG AGG TCG CCT CTA AA 3’ Tm = 58C

Translation: M Q R S P L

Cloning

TAC AAG ATC CGG AAT GCA GAG GTC GCC TCT AAA
Translation: Y K I R N A E V A S K This is wrong!

The CFTR gene has to be in frame with the GFP gene

Design the forward primer to clone CFTR as a GFP fusion 6
This base is the problem

TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Add an extra base!

Forward Primer

5’ ATGC TCCGGA G ATG CAG AGG TCG CCT CTA AA 3’ Tm = 58C

TAC AAG ATC CGG AGA TGC AGA GGT CGC CTC TAA A
This is still wrong!
Translation: Y K I R R C R G R L STOP
Design the forward primer to clone CFTR as a GFP fusion 6
This base is the problem

TAC AAG ATC CGG ACT CAG ATC TCG AGC AAG CTT
BspEI HindIII

Let’s add another one

Forward Primer

5’ ATGC TCCGGA GG ATG CAG AGG TCG CCT CTA AA 3’ Tm = 58C

TAC AAG ATC CGG AGG ATG CAG AGG TCG CCT CTA AA
This is correct!
Translation: Y K I R R M Q R S P L
Design the forward primer to clone CFTR as a GFP fusion 6

7
TACAAGATCCGGACTCAGATCTCGAGCAAGCTT
BspEI HindIII

Cloning
7
TACAAGATCCGGAATGCAGAGGTCGCCTCTAAA Out of frame

vs.
9
TACAAGATCCGGAGGATGCAGAGGTCGCCTCTAAA In frame

Distance between the last codon of GFP and the first codon of the gene must be a multiple of THREE!

If its not, add the minimal number of bases (G is a good choice) to the primer to make it a multiple of three

Add the extra bases between the restriction site and the start of your gene in the primer
What is the maximum number of extra bases you 6
may have to add between the restriction site and
the start of a gene in the primer?
A. 1

B. 2

C. 3

D. 4
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Which forward primer would you use to clone the
follow gene as a N-terminal fusion with GFP?
Vector MCS Gene Sequence:

GFP ATGCCTTCCAATACTCTTCTA …
AAGACAAGCTTCGAATTC
HindIII EcoRI

A. ATGCAAGCTTATGCCTTCCAATACTCTTCTA (Tm=58C)

B. ATGCAAGCTTGGATGCCTTCCAATACTCTTCTA (Tm=58C)

C. ATGCGAAGCTTATGCCTTCCAATACTCTTCTA (Tm=58C)

D. ATGCAAGCTTGATGCCTTCCAATACTCTTCTA (Tm=58C)
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If tagging the C-terminus of CFTR: add extra bases
to the reverse primer and remove stop codon:

C-terminal GFP tag CFTR GFP

CFTR Gene … TTA CCT CTG CCT CAG AGA ACA AGG ATG AAT TAA
Reverse Primer
Stop codon is excluded
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Add extra bases to the reverse primer if tagging
the C-terminus of CFTR and remove stop codon:
13

EcoRI PstI BamHI AgeI

Reverse Primer
CFTR Gene … TTA CCT CTG CCT CAG AGA ACA AGG ATG AAT TAA

Add terminal bases, AgeI site, and two extra bases to 5’ end of primer to put CFTR in frame with GFP (13 + 2 = 15)

Reverse Primer
5’ ATGC ACCGGT GG ATT CAT CCT TGT TCT CTG AG 3’

Final cloned product:

… CTG CCT CAG AGA ACA AGG ATG AAT CCA CCG GTC GCC ACC ATG GTG …
CFTR (no stop) GFP