VECTORS.DOC 01/09/19 Page 1
Explanation of the Smith Lab Phage Display Vectors
Filamentous phage
The filamentous virion [reviewed by (Webster, 2001)] consists of a stretched-out loop of single-stranded 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 host-cell 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.
Feature / Function / PositionsCoding sequences
Gene II / Replication / 87829183828
Gene X / Replication (?) / 496828
Gene V / ssDNA binding / 8431103
Gene VII / Minor coat protein / 11081206
Gene IX / Minor coat protein / 12061301
Gene VIII / Major coat protein
Signal peptide / 13011369
Mature protein / 13701519
Gene III / Minor coat protein
Signal peptide / 15791632
Mature protein / 16332850
Gene VI / Minor coat protein / 28563191
Gene I / Morphogenetic protein / 31974240
Gene IV / Morphogenetic protein / 42215498
TetA / Tetracycline resistance / 62427444
TetR / Repressor of TetA / 75268146
Promoters / 388423
11621196
15071544
40704107
74717506
74737508
87088745
Transcription
terminators / Rho-independent / 15381564
Rho-dependent / 33213344
Rho-dependent / 55005565
Packaging signal / Packaging ssDNA / 55005577
Replication origins / Disrupted minus origin / 56245645,84218532
plus origin / 85418680
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, 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-replication-defective 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.
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:
Vector / Structure of productive insertfUSE1 / 5’-N|NNN|....|NNN|N-3’
3’-N|NNN|....|NNN|N-5’
fUSE2 / 5’-GAT|CNN|.....|NNN-3’
3’-NN|.....|NNN|CTA|G-5’
fUSE3 / 5’-T|CGA|NNN|.....|NNN-3’
3’-NNN|.....|NNN|AGC|T-5’
fUSE5, fUSE55 / 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 wild-type 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:
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 NheI
TAATG AACTCAGATACCCAGCCCGCCTAATGAGCGGGCTTTTTTTT AAGCTAGCTT
A in wt terminator 5980 in fd-tet
should be OK, since
mutation lies outside stem-loop
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 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 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-tet-derived 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 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). Wild-type 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. Replication-defective 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: fd-tet. Gene 9 (1980) 127-140.