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Explanation of the Smith Lab Phage Display Vectors
Filamentous phage
The filamentous virion [reviewed by (Webster, 2001)] consists of a stretched-out loop of singlestranded DNA (ssDNA) sheathed in a tube composed of several thousand copies of the major coat protein
pVIII (product of gene VIII); this protein bears the foreign amino acids in some phage display vectors.
Four minor coat proteins are found at the tips of the virion, pIII (product of phage gene III) being of
particular interest here since it bears foreign amino acids in other phage display vectors. Infection begins
with attachment of pIII to an F pilus, the entering ssDNA being converted to double-stranded replicative
form (RF), which replicates and eventually serves as template for progeny ssDNA. Progeny ssDNA is
extruded through the inner membrane, concomitantly acquiring the coat proteins, some (probably all) of
which are membrane proteins. This process is not lethal to the host: infected cells continue to multiply,
albeit at a slower rate (hence plaque-formation).
The strand of DNA packaged in the virion is called the plus strand; it is anti-complementary to all
the viral mRNA’s. Synthesis of the plus strand occurs by the rolling circle mechanism, beginning at the
plus-strand origin. Synthesis of the other strand—the minus strand—is initiated on plus strands by hostcell RNA polymerase (not primase) at the minus-strand origin. The minus-strand origin is partly optional,
however: the normal host replication machinery, including primase RNA polymerase, can support an
inefficient mode of minus-strand synthesis that starts at more or less random points in the genome.
fd-tet, the parent of all our vectors (GenBank Accession AF317217)
In this article we focus exclusively on our own vectors, which are all based on fd-tet; for other
vector designs the reader is referred to a recent review (Smith and Petrenko, 1997). Phage fd-tet (Zacher
et al., 1980) has the 2775-bp BglII fragment of transposon Tn10 (the complement of positions 632–3406
of GenBank locus TRN10TETR) inserted into the BamHI site of wild-type phage fd (between positions
5645 and 5646 of GenBank locus PFDCG). The resulting chimera has 9183 base pairs. The Tn10
fragment includes a tetracycline resistance determinant consisting of two genes that are inducible by the
antibiotic: the tetA gene, which encodes the resistance protein, is transcribed in the opposite direction to
the phage genes; and the tetR gene, which encodes the repressor of the tetA gene, is transcribed in the
same direction as the phage genes. Because of its Tn10 fragment, fd-tet confers tetracycline resistance on
the host and can be propagated like a plasmid independently of phage function.
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Feature
Coding sequences
Gene II
Gene X
Gene V
Gene VII
Gene IX
Gene VIII
Gene III
Gene VI
Gene I
Gene IV
TetA
TetR
Promoters
Transcription
terminators
Packaging signal
Replication origins
Function
Positions
Replication
Replication (?)
ssDNA binding
Minor coat protein
Minor coat protein
Major coat protein
Signal peptide
Mature protein
Minor coat protein
Signal peptide
Mature protein
Minor coat protein
Morphogenetic protein
Morphogenetic protein
Tetracycline resistance
Repressor of TetA
87829183828
496828
8431103
11081206
12061301
13011369
13701519
15791632
16332850
28563191
31974240
42215498
62427444
75268146
388423
11621196
15071544
40704107
74717506
74737508
87088745
Rho-independent
15381564
Rho-dependent
33213344
Rho-dependent
55005565
Packaging ssDNA
55005577
Disrupted minus origin 56245645,84218532
plus origin
85418680
The low copy number of fd-tet reduces cell killing and has other consequences
The Tn10 fragment insert in fd-tet disrupts the origin of minus-strand synthesis (Smith, 1988).
This greatly reduces the intracellular copy number of the circular, double-stranded replicative form of the
viral DNA (RF), without greatly reducing phage yield.
As a result of their reduced copy number, fd-tet mutants that are completely defective for
assembly nevertheless can be propagated, whereas such mutants in other strains of filamentous phage kill
the host without yielding progeny particles—a phenomenon called “cell killing.” The absence of cell
killing in fd-tet has at least two possible advantages. First, partial defects in coat-protein function due to
insertion of foreign peptides or protein domains should be much better tolerated than in wild-type phage,
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reducing selective pressure for loss or alteration of the insert. Second, it makes possible the frameshifted
fUSE vectors (e.g., fUSE 1, 3 and 5) to be described below.
Even a coat protein defect that is too subtle to induce conspicuous cell killing—for example,
presence of a small displayed peptide—might nevertheless lead to loss of an insert in non-replicationdefective vectors (Smith and Fernandez, 2004).
The replication defect in the fd-tet family of vectors does have two significant drawbacks. First, it
makes RF DNA isolation for construction of libraries somewhat more arduous. CsCl/ethidium bromide
equilibrium density gradient centrifugation is required to give high purity RF DNA (RFmaxiprep.doc),
though RF DNA of sufficient purity for library constructions can be prepared by ion exchange
chromatography on QIAGEN columns (RFmaxiprep2.doc); the yield is only ~200 µg per liter of culture.
Second, the replication defect also reduces infectivity from about 0.5 infectious units per physical particle
for wild-type phage to 0.05–0.2 infectious units per particle for fd-tet.
Because of the interplay of repressor and resistance protein, increasing the number of Tn10
resistance determinants in a cell is reported to actually decrease resistance to the drug. Phage fd-tet
confers a high level of resistance to the drug (at least 40 µg/ml), but the same determinant on a wild-type
(high copy-number) filamentous phage may not confer such strong resistance.
fd-tet is propagated as a tetracycline resistance plasmid and titered as tetracycline
resistance transducing units (TU)
Because of their replication defect, phage fd-tet and its derivatives give tiny plaques, and it is not
practical to titer them as plaque-forming units (pfu). Instead, infective phage are detected as tetracycline
transducing units (TU): that is, cells are infected with the phage and, after a gene-expression period (to
allow the tetracycline resistance protein to be expressed), spread on plates with tetracycline.
fUSE vectors display on all copies of pIII
The fUSE vectors (fUSE1, 2, 3, 5 and 55; (Parmley and Smith, 1988; Scott and Smith, 1990)
(GenBank Accession for fUSE5 is AF218364) are “Type 3” vectors (Smith, 1993; McConnell et al.,
1994). This means there is a single phage chromosome (genome) bearing a single gene III, which accepts
foreign DNA inserts and encodes a single type of pIII molecule. The foreign peptide encoded by the
insert is therefore theoretically displayed on all five pIII molecules on a virion (though in practice normal
proteolytic enzymes in the host bacterium often remove the foreign peptide from some or even most
copies of pIII, especially if the foreign peptide is large). The only fUSE vector we distribute regularly is
fUSE55; fUSE2 and fUSE3 may be supplied on special request; fUSE1, which is exceedingly difficult to
work with, is no longer available.
The nucleotide sequence of the plus strand of the fd-tet parent and of four fUSE vectors in the
vicinity of the cloning site(s) are shown in the table below (fUSE5’s successor fUSE55 will be described
below). The fd-tet sequence shown corresponds to positions 1630–1650 of the vector. The first codon in
each sequence encodes the last amino acid (Ser) of the pIII signal peptide; the second codon therefore
programs the first amino acid of the mature form of pIII—the form in the secreted virion.
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PvuII
fUSE1 5’-TCC GCT --- --- --- --- --- --- --- --- GAC AGCT GTT
XhoI
fUSE3 5’-TCC GCT --- --- --- --- --- --- --- --- GAA A-CT --SfiI
SfiI
fUSE5 5’-TCG GCC GAC GTG GCC TGG CCT CTG GGG CC- GAA A-CT GTT
BglII
fUSE2 5’-TCC GCT --- --- --- --- --- --- --- --- GAA G-AT CTT
fd-tet
pIII
GAA AGT-3’
-CG AGT-3’
GAA AGT-3’
GAA AGT-3’
5’-TCC GCT --- --- --- --- --- --- --- --- GAA A-CT GTT GAA AGT-3’
Ser Ala - Glu Thr Val Glu Ser
Signal
peptidase
The fUSE1, fUSE2 and fUSE3 vectors have a single cloning site, as indicated above. There are
two SfiI cloning sites in fUSE5 (and its successor fUSE55; see next paragraph), which have non-identical,
non-complementary 3-base 3´ overhanging ends; this allows directional cloning after removal of the
stuffer that lies between these sites in the vector.
Vector fUSE55 supersedes fUSE5 (which is no longer available) and can be used in place of
fUSE5 in all our previous protocols. It has two changes relative to fUSE5:

The 14-bp stuffer sequence is changed to TGGCCCGGCCTCTG in order to remove a dcm
methylation site

A BglI site in tetA (the gene encoding the tetracycline resistance protein) is removed
without changing the amino acid sequence); the two SfiI sites are also the only two BglI sites in
the fUSE55 vector, so that BglI can be used in place of SfiI for cloning.
The gene-III reading frame is disrupted in fUSE1, fUSE3, fUSE5 and fUSE55, abolishing all pIII
functions, including infectivity. The vectors are nevertheless propagatable as tetracycline-resistance
plasmids because of the absence of cell killing in fd-tet. If an insert spliced into these vectors restores the
gene-III reading frame without stop codons, it also restores infectivity; such frame-restoring inserts, as
well as the clones harboring them, will be called productive (fUSE1, fUSE3, fUSE5 and fUSE55
themselves are nonproductive). Vector fUSE2 has a single BglII cloning site that does not disrupt the
gene-III reading frame. Some of the fragments cloned into that site will preserve the gene-III reading
frame without stop codons and thus be productive. The general structure of productive inserts for each of
the vectors is indicated in the following table, the plus strand being shown on top and the reading frame
being indicated by vertical bars:
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Vector
fUSE1
fUSE2
fUSE3
fUSE5,
fUSE55
Structure of productive insert
5’-N|NNN|....|NNN|N-3’
3’-N|NNN|....|NNN|N-5’
5’-GAT|CNN|.....|NNN-3’
3’-NN|.....|NNN|CTA|G-5’
5’-T|CGA|NNN|.....|NNN-3’
3’-NNN|.....|NNN|AGC|T-5’
5’-NN|.....|NCT|G-3’
3’-TG|CNN|.....|N-5’
Nonproductive fUSE phage are noninfective and therefore give no transductants; they make no
contribution to the library, since clones are ultimately detected as TU. This eliminates the background of
nonproductive clones, including clones without inserts in the frame-shifted vectors fUSE1, fUSE3, fUSE5
and fUSE55.
f88 vectors display on ~150 copies of pVIII
The f88 vectors (including f88-4; GenBank Accession AF218363) are Type 88 vectors, in which
the phage genome bears two genes VIII, encoding two different types of pVIII molecule. One pVIII is
recombinant (i.e., bears a foreign DNA insert) and the other wild-type. The recombinant gene VIII is
synthetic and differs in nucleotide sequence from the wild-type gene (though it largely encodes the wildtype amino acid sequence). The f88 virion is a mosaic, its coat being composed of both wild-type and
recombinant pVIII subunits; the latter typically comprise about 150 of the 3900 subunits. This allows
hybrid pVIII proteins with quite large foreign peptides to be displayed on the virion surface, even though
the hybrid protein by itself cannot support phage assembly.
Here is the sequence of the recombinant gene VIII of vector f88-4 (GenBank Accession
AF218363), whose total genome length is 9234 base-pairs:
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5647 in fd-tet
 XhoI
AGCTCGAGCTTACTCCCCA
tac promoter
TCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTC
gene VIII translation initiation region
TTAATGGAAACTTCCTC ATG AAA AAG TCT TTA
M
K
K
S
L
GTT CTT AAA GCA TCT GTT GCT GTT GCG ACT CTT GTT
V
L
K
A
S
V
A
V
A
T
L
V
HindIII
PstI

stuffer

CCT ATG CTA^AGC TTT GCC AAC GTC CCT GCA^GAA GGT GAT GAC CCG GCT AAA
P
M
L
S
F
A ^ N
V
P
A
E
G
D
D
P
A
K
Signal Peptidase
GCT GCT TTT GAC TCT CTT CAG GCT TCT GCT ACT GAA TAC ATC GGC TAC GCT
A
A
F
D
S
L
Q
A
S
A
T
E
Y
I
G
Y
A
TGG GCT ATG GTG GTT GTT ATC GTT GGT GCT ACT ATT GGC ATC AAA CTT TTC
W
A
M
V
V
V
I
V
G
A
T
I
G
I
K
L
F
AAA AAA TTC ACT TCT AAA GCG TCT
K
K
F
T
S
K
A
S
trpA terminator
AACTCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT

A in wt terminator
should be OK, since
mutation lies outside stem-loop
TAATG
NheI
AAGCTAGCTT

5980 in fd-tet
Foreign inserts are spliced between the HindIII and PstI cloning sites after removing the stuffer
that lies between them in the vector. Productive inserts have the following general structure:
5´-AGC|TTT|GCC|NNN|...|NNT|GCA-3´
3´-AA|CGG|NNN|...|NN-5´
Again, the plus strand is shown on top and the reading frame is indicated by the vertical bars. The first
three codons of the plus (top) strand encode the last three amino acids of the signal peptide: –Ser-Phe-Ala.
Because the recombinant gene VIII is transcribed from a tac promoter, full expression in a lacIQ
strain like K91BluKan (and also perhaps in a lacI+ strain, since f88-4 is a low-copy-number replicon)
requires an inducer like IPTG (1 mM works well) and absence of glucose. In a lacI strain like MC1061,
however, the IPTG is not necessary. The percent of pVIII molecules that are recombinant in fully
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inducing conditions ranges from a few percent to ~20%, as estimated by gel electrophoresis (see the
document entitled p8page.doc).
The f8 vectors display on all 3900 copies of pVIII
The f8 vector f8-1 (GenBank Accession AF218734) is a Type 8 vector, which displays foreign
peptides on every copy of the major coat protein pVIII. Only short foreign peptides can be displayed on
every copy of pVIII; even so, the peptide comprises a substantial fraction of the virion’s mass, and can
dramatically alter its physical and biological properties (Kishchenko et al., 1991; Kishchenko et al., 1994;
Petrenko et al., 1996). Here is the nucleotide sequence:
PstI
BamHI
Viral DNA —GCT GCA GAG GGT GAG GAT CCC—
Mature pVIII
Ala Glu Gly Glu Asp Pro—
1
2
3
4
5
6
The f8 vectors are highly specialized, and not suitable for most phage display applications. To learn
about these vectors and the libraries derived from them, read the basic reference (Petrenko et al., 1996)
and contact Valery Petrenko at [email protected] if you’re still interested.
fd-cat confers resistance to chloramphenicol rather than tetracycline
Phage fd-cat has a 7775-base genome in which most of the tetracycline resistance determinant of
fd-tet is replaced with the chloramphenicol acetyl transferase (cat) gene from plasmid vector pBR328. It
has the same replication defect as fd-tet; its infectivity is roughly 5 times less on average (typically ~1%).
This phage has the same virion proteins as fd-tet, and as the library phage without their displayed
peptides; it is used as an internal enrichment control during affinity selection, as described in
AffinitySelection.doc.
Beware of wild-type “freeloaders”
If a culture of fd-tet-derived phage (with the replication defect) in infectable host cells becomes
contaminated with “wild-type” phage (i.e., any filamentous phage without the replication defect,
including the popular M13mp series vectors), the wild-type virions can come to dominate the culture—
even if the culture medium contains tetracycline and the wild-type contaminant does not bear a
tetracycline resistance determinant. The reason is that when a cell is co-infected with both the fd-tetderived phage (conferring resistance to tetracycline) and a wild-type phage, the latter (the “freeloader”)
out-replicates the former and thus dominates the population of virions secreted by that cell.
Coinfection is an ongoing process. That’s because a cell harboring a replication-defective phage
will occasionally segregate a daughter cell with no phage genome. The phage-less cell is genotypically
sensitive to tetracycline, but remains phenotypically resistant for plenty enough time to regenerate its F
pilus and thus regain the ability to be infected (or coinfected) by new phage (chronically infected cells
lose their F pili and thus can’t be super-infected). This ongoing cycle of segregation of phage-less
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daughters followed by coinfection provides a way for an initially minor wild-type contaminant to take
over a culture.
Presence of wild-type contaminants can be assayed by titering for plaques (Plaques.doc). Wildtype phage make large plaques, while fd-tet-derived phage make very tiny (often invisible) plaques as a
result of their replication defect.
Theoretically, at least, even a replication-defective phage without a tetracycline resistance
determinant (e.g., fd-cat phage; previous subsection) could act as a freeloader. However, it’s unlikely that
such a phage could come to dominate a culture if were initially a minor contaminant. Replicationdefective freeloaders are therefore of much less concern than wild-type freeloaders.
References
Kishchenko, G., Batliwala, H. and Makowski, L.: Structure of a foreign peptide displayed on the surface
of bacteriophage M13. J Mol Biol 241 (1994) 208-13.
Kishchenko, G.P., Minenkova, O.O., Ilyichev, A.A., Gruzdev, A.D. and Petrenko, V.A.: Study of the
structure of phage-M13 virions containing chimeric B-protein molecules. Mol. Biol.-Engl. Tr. 25
(1991) 1171-1176.
McConnell, S.J., Kendall, M.L., Reilly, T.M. and Hoess, R.H.: Constrained peptide libraries as a tool for
finding mimotopes. Gene 151 (1994) 115-8.
Parmley, S.F. and Smith, G.P.: Antibody-selectable filamentous fd phage vectors: affinity purification of
target genes. Gene 73 (1988) 305-318.
Petrenko, V.A., Smith, G.P., Gong, X. and Quinn, T.: A library of organic landscapes on filamentous
phage. Protein Engineering 9 (1996) 797-801.
Scott, J.K. and Smith, G.P.: Searching for peptide ligands with an epitope library. Science 249 (1990)
386-90.
Smith, G.P.: Filamentous phage assembly: morphogenetically defective mutants that do not kill the host.
Virology 167 (1988) 156-65.
Smith, G.P.: Preface. Surface display and peptide libraries. Gene 128 (1993) 1-2.
Smith, G.P. and Fernandez, A.M. Effect of DNA copy number on genetic stability of phage-displayed
peptides. Biotechniques 36 (2004) 610-4, 616, 618.
Smith, G.P. and Petrenko, V.A.: Phage Display. Chemical Reviews 97 (1997) 391-410.
Webster, R.: Filamentous phage biology. In: Barbas III, C.F., Burton, D.R., Scott, J.K. and Silverman,
G.J. (Eds.), Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 2001, pp. 1.1-1.37.
Zacher, A.N.I., Stock, C.A., Golden, J.W.I. and Smith, G.P.: A new filamentous phage cloning vector: fdtet. Gene 9 (1980) 127-140.