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The Smith Lab Phage Display Vectors
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This document provides detailed information on the fd-tet-based bacteriophage vectors in the phage display system developed by George P. Smith at the University of Missouri. Please visit the website, Smith Lab Website, for more information.
Procedure
Filamentous Bacteriophage
The filamentous bacteriophage virion (see Citation #1, Citation #2, and Ciation #3) consists of a stretched-out loop of single-stranded DNA (ssDNA) sheathed in several thousand copies of the major coat protein pVIII (the product of phage gene VIII). pIII (the product of phage gene III) is one of four minor coat proteins that localize to the tips of the virion, with up to five molecules of pIII per tip. In this phage display system, vectors encoding pIII and pVIII have been engineered to display foreign amino acids in a recombinant library.
Infection begins with the attachment of a bacteriophage virion to the F pilus of a bacterium. The pIII protein mediates this attachment. Phage genomic ssDNA enters the bacterial cell and is converted by host cell enzymes to double-stranded replicative form (RF) DNA. RF DNA replicates episomally and serves as a template for the production of progeny ssDNA (see Hint #1). To complete the bacteriophage life cycle, newly synthesized progeny ssDNA molecules are extruded through the inner membrane of the infected host and packaged into phage particles, acquiring a lipid bilayer and coat proteins as the particles pass through the membrane. This process is not lethal to the host and infected cells continue to multiply, albeit at a slower rate.
fd-tet, the Parent of the Vectors in This Phage Display System (see Hint #2)
Phage fd-tet (see Citation #4) has a 2775 basepair BglII fragment of transposon Tn10 inserted into the BamHI site of the wild-type phage fd genome. The resulting recombinant molecule is 9183 base pairs in size (see Hint #3). The Tn10 fragment includes a tetracycline resistance determinant consisting of two genes that are inducible by the antibiotic (see Hint #4). Because of the Tn10 fragment, infection with fd-tet confers tetracycline resistance to the host and can be propagated like a plasmid independent of phage function.
Advantages of the Low Copy Number of fd-tet
The Tn10 insertion in fd-tet disrupts the minus-strand origin of replication (see Citation #5). This greatly reduces the intracellular copy number of the circular, double-stranded RF DNA without greatly reducing phage yield. As a result, fd-tet mutants that are completely defective for assembly are still propagated, whereas such mutants in other strains of filamentous phage in which Tn10 has been inserted at different loci in the fd genome kill the host without yielding progeny particles, a phenomenon known as "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, the absence of cell killing enables the use of the frameshifted fUSE vectors (e.g., fUSE 1, 3 and 5) described below.
The low copy number does have disadvantages. Larger cultures of infected bacteria and purification by Cesium Chloride centrifugation are required for the isolation of sufficient amounts of phage DNA for library construction (see Protocol ID #2170). Infectivity is also reduced from 0.5 infectious units per physical particle for wild-type fd phage to approximately 0.05 to 0.2 infectious units per particle for fd-tet phage.
Titering fd-tet Infectivity
In general, the infectivity of viruses (titer) is measured as plaque-forming units (pfu) on a lawn of infectible host cells. To measure pfu's, serial dilutions of viral particles are incubated with infectible cells before plating on solid media. A count of the number of circular regions of growth inhibition on a lawn of cells and the corresponding dilution factor will determine the titer of the viral particles (usually expressed as pfu/ml; see Protocol ID #2181).
Because of the replication defect resulting from the Tn10 insertion, phage fd-tet and its derivatives yield tiny plaques such that it is not practical to titer them as plaque-forming units. Instead, the infectivity of the mutant phage is measured as tetracycline transducing units (TU). fd-tet RF DNA is propagated as a tetracycline resistance plasmid and titered by measuring the resistance of infected cells to the antibiotic on solid medium (see Protocol ID #2173).
fUSE Vectors: Display on pIII
The fUSE vectors (fUSE1, 2, 3 and 5) are "Type 3" vectors (see Citation #6 and Citation #7). This designation refers to a modification of the filamentous phage genome to contain a single phage chromosome bearing a single gene III. Foreign DNA is inserted into the gene III sequence, resulting in the production of recombinant pIII molecules displaying foreign peptides. A foreign peptide encoded by an insert is therefore theoretically displayed on all five pIII molecules on a virion (see Hint #5).
The nucleotide sequence of the plus-strand of the fd-tet parent and of the four fUSE vectors in the vicinity of the cloning site(s) are shown in Image #1A. The fd-tet sequence shown corresponds to positions 1630 through 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, secreted form of pIII present on virus particles.
The fUSE1, fUSE2 and fUSE3 vectors have a single restriction enzyme recognition site for cloning inserts (PvuII, XhoI, and BglII, respectively), as indicated in Image #1A. The fUSE5 vector contains two SfiI cloning sites, and digestion with SfiI results in non-identical, non-complementary 3' overhangs of three nucleotides. This allows for directional cloning of inserts after the vector is purified away from the "stuffer" fragment released by digestion.
The gene III open reading frame is disrupted in fUSE1, fUSE3 and fUSE5 vectors, abolishing infectivity (pIII protein is required for attachment of the virion to the F pilus). The vectors are still propagated in cells as tetracycline-resistance plasmids because of the absence of cell killing. If an insert cloned into these vectors restores the gene III reading frame without introducing stop codons, infectivity is restored. Such frame-restoring inserts, and the clones harboring them, are termed productive (fUSE1, fUSE3 and fUSE5 without insert are nonproductive). Vector fUSE2 has a single BglII cloning site that does not disrupt the reading frame of gene III and is therefore productive without insert. The general structure of productive inserts for each of the vectors is indicated in Image #1B. The plus-strand is shown on top and the reading frame is indicated by vertical bars (see Image #1B).
Nonproductive fUSE phage are unable to infect bacteria and therefore are not propagated. They make no contribution to a recombinant library, since clones are ultimately detected as transducing units (see Protocol ID #2173). This eliminates the background of nonproductive clones, including those clones without inserts in the frame-shifted vectors fUSE1, fUSE3 and fUSE5.
f88 Vectors: Display on pVIII
The f88 vectors (including f88-4) are "Type 88" vectors. This designation refers to a modification of the filamentous phage genome to contain two copies of gene VIII. One copy is an original wild type gene VIII. The other copy bears a restriction enzyme recognition site into which foreign DNA is inserted, resulting in the production of recombinant pVIII molecules displaying foreign peptides. The recombinant gene VIII is synthetic and differs in nucleotide sequence from the wild type copy. Both copies of gene VIII are expressed, resulting in a mosaic virion possessing a protein coat composed of both wild type and recombinant pVIII subunits. The recombinant molecules typically comprise about 150 of the 3900 subunits. The low abundance of the recombinant molecules relative to the wild type ones permits large foreign peptides to be displayed on the virion surface, even though the recombinant molecule by itself cannot support phage assembly.
The sequence and features of the recombinant gene VIII of vector f88-4, whose total genome length is 9234 base-pairs, is depicted in Image #1C. Foreign DNA is directionally cloned into the HindIII and PstI restriction enzyme recognition sites after removal of the "stuffer" fragment from the vector. Productive inserts have the general structure as indicated in Image #1D. 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-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 exposure of the cells to an inducer like Isopropylthio-β-D-galactoside (IPTG; 1 mM IPTG in growth medium works well) and absence of glucose. In a ΔlacI strain like MC1061, however, the IPTG is not necessary. The percent of pVIII molecules coating a virion that are recombinant in fully inducing conditions ranges from a few percent to about 20 percent, as estimated by gel electrophoresis.
f8 Vectors: Display on All pVIII Molecules
The vector f8-1 is a "Type 8" vector, which displays foreign peptides on all 3900 coat protein pVIII molecules of a recombinant virion. Only short peptides can be displayed on pVIII molecules and the foreign peptides comprise a substantial fraction of the mass of the virion. This can dramatically alter the physical and biological properties of the virion (see Citation #8 and Citation #9). The nucleotide sequence is depicted in Image #1E (see Hint #6).
Solutions
This bioProtocol does not use any solutions
BioReagents and Chemicals
This bioProtocol does not use any reagents
Protocol Hints
1. The strand of ssDNA packaged in the virion is called the plus strand. It is anti-complementary to viral messenger RNA (mRNA). Synthesis of the plus strand occurs by the "rolling circle" mechanism, beginning at the plus-strand origin of replication. Synthesis of the minus-strand is initiated on plus-strands by host-cell RNA polymerase (not primase) at the minus-strand origin of replication. The minus-strand origin of replication is not required for replication; the normal host replication machinery, which employs RNA polymerase primase, can support an inefficient mode of minus-strand synthesis that starts at more or less random points in the genome.
2. For other vector designs the contributor of this document references Citation #3.
3. The sequence of the transposon Tn10 BglII fragment is the complement of nucleotides 632-3406 of the GenBank locus, TRN10TETR. To access this sequence, visit the following link, Entrez-Nucleotide, and enter TRN10TETR in the search window. The insertion site in the wild type fd genome can be found between nucleotides 5645 and 5646 of the GenBank locus PFDCG. For the complete sequence of fd-tet and a table of features, follow this link: Sequence Features.
4. As inserted, the tetA gene, which encodes the tetracycline 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. The interplay of the repressor and resistance proteins is important in determining the level of resistance to the antibiotic. Increasing the number of Tn10 resistance determinants in a cell is reported to actually decrease resistance to the drug. Thus, phage fd-tet confers a high level of resistance to tetracycline (at least 40 μg/ml), but the same determinant on a wild-type (high copy-number) filamentous phage may not confer such strong resistance.
5. 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.
6. The f8 vectors are highly specialized, and not routinely used. To obtain these vectors, read the basic reference (see Citation #10) and contact The Smith Lab Website.
Citation and/or Web Resources
3. Fulford W, Model P. Bacteriophage f1 DNA replication genes. II. The roles of gene V protein and gene II protein in complementary strand synthesis. (1988) J Mol Biol. 203(1): 39-48.
10. Petrenko, V. A library of organic landscapes on filamentous phage. (1996) Protein Eng Sep 9:9: 797-801.
2. Fulford W, Model P. Regulation of bacteriophage f1 DNA replication. I. New functions for genes II and X. (1988) J Mol Biol. 203(1): 49-62.
4. Zacher AN 3d, Stock CA, Golden JW 2d, Smith GP. A new filamentous phage cloning vector: fd-tet. (1980) Gene 9:1-2:127-40
9. Kishchenko G, Batliwala H, Makowski L. Structure of a foreign peptide displayed on the surface of bacteriophage M13. (1994) J Mol Biol 241:2: 208-13.
1. Marvin DA, Hale RD, Nave C, Citterich MH. Molecular models and structural comparisons of Native and mutant class I filamentous bacteriophages Ff (fd, f1, M13), If1 and IKe. (1994) J Mol Biol. 235(1): 260-86.
8. Kishchenko GP, Minenkova OO, Il'ichev AI, Gruzdev AD, Petrenko VA. Structure of virions of the M13 phage containing chimeric B-protein molecules. (1991) Mol Biol (Mosk) 25:6: 1497-1503.
7. Smith GP, Scott JK. Libraries of peptides and proteins displayed on filamentous phage. (1993) Methods Enzymol. 217: 228-57.
5. Smith GP. Filamentous phage assembly: morphogenetically defective mutants that do not kill the host. (1988) Virology 167(1): 156-65.
6. Parmley SF, Smith GP. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. (1988) Gene 73(2): 305-18.
