The Function and Organization of Plasmids

1. Introduction

In 1952, Joshua Lederberg coined the term plasmid to describe any bacterial genetic element that exists in an extrachromosomal state for at least part of its replication cycle . As this description included bacterial viruses, the definition of what constitutes a plasmid was subsequently refined to describe exclusively or predominantly extrachromosomal genetic elements that replicate autonomously. ( ...)

. 0

Basic Plasmid Characteristics Size and Copy Number

Naturally occurring plasmids vary greatly in their physical properties, a few examples of which are shown in Table 1. They range in size from <2-kilobase pair (kbp) plas-mids, which can be considered to be elements simply capable of replication, to From: Methods in Molecular Biology, Vol. ( ...)

. 1

Geometry

Although most plasmids possess a circular geometry, there are now many examples in a variety of bacteria of plasmids that are linear (15,16). As linear plasmids require specialized mechanisms to replicate their ends, which circular plasmids and chromosomes do not, linear plasmids tend to exist in bacteria that also have linear chromosomes (17). ( ...)

. 2

Plasmid-Encoded Traits

Many plasmids are phenotypically cryptic and provide no obvious benefit to their bacterial host other than the possible exclusion of plasmids that are incompatible with the resident plasmid (see Part 2). ( ...)

. 3

Plasmid Replication

Plasmids, like chromosomes, are replicated during the bacterial cell cycle so that the new cells can each be provided with at least one plasmid copy at cell division (41). To this end, plasmids have developed a number of strategies to initiate DNA replication but have mostly co-opted the host polymerization machinery (42) for subsequent stages of DNA synthesis, thereby minimizing the amount of plasmid-encoded information required for their replication. ( ...)

. 4

Iteron-Containing Replicons

The genetic organization of a stylized plasmid replicon is illustrated in Fig. 2A. This replicon consists of a number of elements, including a gene for a plasmid-specific replication initiation protein (Rep), a series of directly repeated sequences (iterons), DnaA boxes, and an adjacent AT-rich region. ( ...)

. 5 6

ColEI-Type Replicons

The replicon of the ColEl plasmid of Escherichia coli is the basis for many gene-cloning and gene-expression vectors that are commonly used in current molecular biology (see Parts 2 and 28). In contrast to the replication of iteron-containing plasmids, ColE1 replication proceeds without a plasmid-encoded replication initiation protein and instead utilizes an RNA species in initiation and RNA-RNA interactions to achieve copy number control (see Fig. ( ...)

. 7

Rolling-Circle Replication

Many small (<10 kbp) plasmids of Gram-positive Eubacteria replicate by a rolling-circle mechanism, which is distinct from the replication of iteron-containing or ColE1-like plasmids (see Fig. 3) (47). ( ...)

. 8

Plasmid Segregation

DNA replication produces precise plasmid copies, but plasmids must also ensure that they are distributed to both daughter cells during bacterial cell division. If the Fig. 3. Replication of rolling-circle plasmids. ( ...)

. 9

Active Partition Systems

Following plasmid replication, active partitioning systems position the plasmids appropriately within the cell such that at cell division, each of the new cells acquires at least one copy of the plasmid (see Fig. ( ...)

. 10

Site-Specific Recombination

Many laboratory strains of E. coli have been mutated to be deficient in homologous recombination. This reduces the frequency with which genes cloned in multicopy plas-mids undergo rearrangements in these strains. ( ...)

. 11 12

Toxin-Antitoxin Systems

An additional mechanism which plasmids use to favor their maintenance in bacterial populations involves the killing or growth impairment of cells that fail to acquire a copy of the plasmid. This has variously been referred to as postsegregational cell killing, plasmid addiction, or toxin-antitoxin systems (57-60). ( ...)

. 13

Plasmid Dissemination in Bacterial Populations

Certain bacterial species can achieve a state of natural competence for the uptake of naked plasmid DNA (transformation) (62), or can acquire DNA that has been packaged into a bacteriophage head and is injected into the host (transduction) (63). ( ...)

. 14 15

Plasmid Evolution: Plasmids Are Modular Elements

Whole genome and plasmid-specific sequencing projects have recently begun to provide fascinating glimpses into the genetic organization and evolution of plasmids. These studies have revealed that plasmids, particularly large plasmids, are commonly constructed in a modular fashion by the recombination activities of transposons, insertion sequences, bacteriophages, and smaller plasmids (72). ( ...)

. 16 17

Choosing a Cloning Vector

Introduction

Since the construction of the first generation of general cloning vectors in the early 1970s, the number of plasmids created has increased to an almost countless number. Thus, a critical decision facing today's investigator is that of which plasmid to use in a particular project? ( ...)

. 18

Criteria for Choosing a Cloning Vector Insert Size

For projects in which it is desired that a particular piece of DNA be cloned, one consideration is the size of the insert DNA. Most general cloning plasmids can carry a DNA insert up to around 15 kb in size. ( ...)

. 19

Cosmids

Cosmids are conventional vectors that contain a small region of bacteriophage X DNA containing the cohesive end site (cos). This contains all of the cis-acting elements for packaging of viral DNA into X particles. ( ...)

. 20

X Vectors

The bacteriophage X genome comprises 48,502 bp. On entering the host cell, the phage adopts one of two life cycles: lytic growth or lysogeny. In lytic growth, approx 100 new virions are synthesized and packaged before lysing the host cell, releasing the progeny phage to infect new hosts. ( ...)

. 21

Bacterial Artificial Chromosomes

Bacterial artificial chromosomes (BACs) are circular DNA molecules. They contain a replicon that is based on the F factor comprising oriS and repE encoding an ATP-driven helicase along with parA, parB, and parC to facilitate accurate partitioning (see Part 1). ( ...)

. 22

Copy Number

Different cloning vectors are maintained at different copy numbers, dependent on the replicon of the plasmid (see Part 1). In a majority of cases in which a piece of DNA is cloned for maintenance and amplification for subsequent manipulation, the greater the yield of recombinant plasmid from E. ( ...)

. 23

Incompatibility

Incompatibility refers to the fact that different plasmids are sometimes unable to coexist in the same cell. This occurs if the two different plasmids share functions required for replication and/or partitioning into daughter cells. ( ...)

. 24

Selectable Marker

Introduction of plasmids in to E. coli cells is an inefficient process. Thus, a method of selecting those cells that have received a plasmid is required. Furthermore, cells that do not contain a plasmid are at a growth advantage over those that do and, thus, have to replicate both the chromosome and additional plasmid DNA. ( ...)

. 25 26

Ampicillin

This drug inhibits the bacterial transpeptidase involved in peptidoglycan biosynthesis and thus inhibits cell wall biosynthesis (14). As such, ampicillin inhibits log-phase bacteria but not those in a stationary phase. ( ...)

. 27

Kanamycin

A member of the aminoglycoside family of antibiotics, kanamycin was first isolated from Streptomyces kanamyceticus in Japan in 1957. This polycation is taken into the bacterial cell through outer-membrane pores but crosses the cytoplasmic membrane in an energy-dependent process utilizing the membrane potential. ( ...)

. 28

Chloramphenicol

First isolated from a soil actinomycete in 1947, chloramphenicol was widely used as a broad-spectrum antibiotic although its clinical use has been curtailed because of drug-induced bone-marrow toxicity and the emergence of bacterial chloramphenicol resistance. ( ...)

. 29

Tetracycline

Originally isolated from Streptomyces aureofaciens in 1948, there are now many tetracycline derivatives available. They bind to a single site on the 30S ribosomal subunit to block the attachment of aminoacyl tRNA to the acceptor site and thus inhibit protein synthesis (19). ( ...)

. 30

Cloning Sites

The cloning of DNA into a vector usually involves ligation of the insert DNA fragment to vector DNA that has been cut with a restriction endonuclease. This is facilitated by the insert and vector DNA fragments having compatible cohesive ends. ( ...)

. 31

Specialized Plasmid Functions

Some projects will involve specific downstream applications that will require specialized plasmid functions that are only present on some plasmids. For example, both the pUC and pBluescript series of vectors are high-copy-number, ampicillin-resis-tance-conferring plasmids that contain MCSs that facilitate the use of a wide range of restriction endonucleases in the cloning step. ( ...)

. 32

Summary

When choosing a cloning vector for use in a cloning project, the investigator is faced with an enormous choice. However, the application of a small number of criteria can quickly guide the selection of a suitable vector. ( ...)

. 33

Escherichia coli Host Strains

Introduction

To successfully perform molecular genetic techniques it is essential to have a full understanding of the properties of the various Escherichia coli host strains commonly used for the propagation and manipulation of recombinant DNA. ( ...)

. 34

Genotype Nomenclature

A genotype indicates the genetic state of the DNA in an organism. It is associated with an observed behavior called the phenotype. Genotypes of E. coli strains are described in accordance with a standard nomenclature proposed by Demerec et al. ( ...)

. 35

General Properties of Cloning Hosts

The genotypes and features of a representative selection of popular host strains used for general recombinant DNA cloning procedures are listed in Table 2. An extended listing of available strain genotypes can be found in ref. ( ...)

. 36

Disablement

Many laboratory E. coli strains carry mutations that reduce their viability in the wild and preclude survival in the intestinal tract (6). These often confer auxtrophy, that is, they disable the cell's ability to synthesize a critical metabolite, which, therefore, must be supplied in the medium. ( ...)

. 37

Suppressor Mutations

Some vectors contain nonsense mutations in essential genes as a means of preventing spread to natural bacterial populations. Nonsense mutations are chain-termination codons; they are termed amber (UAG) or ochre (UAA) mutations (5). ( ...)

. 38

Fertility Status

Some E. coli strains carry an F episome or fertility factor, which can be found in several different forms (7). It may be carried as a double-stranded single-copy circular extrachromosomal plasmid, designated F+, or if it harbors additional genes, F'. ( ...)

. 39

Restriction and Modification Systems

Restriction-modification systems play a role in preventing genetic exchange between groups of bacteria by enabling the host to recognize and destroy foreign DNA. An archetypal system consists of a DNA methylase and its cognate restriction endonu-clease. ( ...)

. 40

Dam and Dcm Methylation

Derivatives of E. coli K-12 normally contain three site-specific DNA methylases: Dam, Dcm and EcoK. DNA adenine methylase, encoded by dam, methylates adenine residues in the sequence GATC (9,10). ( ...)

. 41

EcoK System

The E. coli K-12 EcoK methylase modifies the indicated adenine residues of the target sequence A(mA)CN6GTGC, and its complement GC(mA)CN6GTT (8,16). The cognate endonuclease will cleave DNA that is unmodified at this sequence. ( ...)

. 42

McrA, McrBC, and Mrr Restriction

E. coli K-12 also contains several methylation-dependent restriction systems, namely McrA, McrBC, and Mrr. The methylcytosine restricting endonucleases, McrA and McrBC, cleave methylcytosines in the sequences CG and (A/C)G, respectively (1821). ( ...)

. 43

Recombination

Following successful transformation of a plasmid vector into E. coli, host recombination systems can catalyze rearrangement of the recombinant molecule. This is a particular problem when the cloned DNA contains direct or inverted repeats and can result in duplications, inversions, or deletions. ( ...)

. 44 45

Recombination Systems in X-Infected Hosts

Bacteriophage X is injected into the E. coli host as a linear molecule that rapidly circularizes and, during the early phase of infection, replicates by a bidirectional 9-type mechanism, yielding monomeric circles. ( ...)

. 46

Complementation

Many current molecular biology techniques rely on the pioneering studies of the lac operon by Jacob and Monod in the 1960s (46). The lac operon consists of three genes: lacZYA, encoding p-galactosidase, which cleaves lactose to glucose and galac-tose, a permease, and a transacetylase. ( ...)

. 47

Hosts for Mutagenesis

The frequency of spontaneous mutation in E. coli may be increased by three to four orders of magnitude by mutations in mutD, which encodes the 3'5' exonuclease sub-unit of the DNA polymerase III holoenzyme (50,51). ( ...)

. 48

Specialized Strains for Protein Expression

E. coli is a popular host for the overexpression of recombinant proteins (see Parts 28 and 29). There are a number of factors that can influence protein yields and careful strain choice can greatly improve the chance of successful expression. ( ...)

. 49

Repressors

E. coli expression vectors utilize highly active inducible promoters and the correct host strain must be used to ensure proper tight regulation (53). Many common vectors Table 4 Properties of E. ( ...)

. 50

Stability

Host proteases can interfere with the isolation of intact recombinant proteins; degradation may be avoided by the use of protease-deficient hosts. In E. coli, lon encodes a major ATP-dependent protease and strains that contain deletions of this gene greatly improve the yield of many recombinant proteins (54,55). ( ...)

. 51

Codon Bias

The frequency with which amino acid codons are utilized varies between organisms and is reflected by the abundance of the cognate tRNA species. This codon bias can have a significant impact on heterologous protein expression, so that genes that contain a high proportion of rare codons are poorly expressed (61,62). ( ...)

. 52

Solubility and Posttranslational Processing

Overproduction of heterologous proteins in E. coli often results in misfolding and segregation into insoluble inclusion bodies. The cytoplasmic chaperones, DnaK-DnaJ and GroES-GroEL, assist proper folding in wild-type E. ( ...)

. 53

Conclusion

Since the first mutants of E. coli K-12 were isolated in the 1940s, laboratory strains have been heavily mutagenized by treatment with X-rays, ultraviolet irradiation, and nitrogen mustard. Thus, they may carry unidentified mutations and it can be useful to try more than one strain background if experiments are unsuccessful. ( ...)

. 54

Chemical Transformation of E. coli

Introduction

Transformation is defined as the transfer of genetic information into a recipient bacterium using naked DNA, without any requirement for contact with a donor bacterium. The ability to transform or accept exogenous DNA is generally referred to as competence, although the term has been so widely used in different systems that it is difficult to generate an all-inclusive definition for competence. ( ...)

. 55

Materials

Preparation of Competent Cells Classical Calcium Chloride Method 1. Host bacterial strain (see Note 1). 2. Luria-Bertani (LB) broth: 5 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl. ( ...)

. 56

Methods

- Preparation of Competent Cells - Classical Calcium Chloride Method (2,3) This method was the first generally applicable method for transformation of E. coli with plasmid DNA with typical yields of 1 x 107 transformants per microgram of DNA and is still in wide use. ( ...)

. 57 58

Notes

1. For factors affecting the choice of host strain, see Part 3. 2. Place solution on ice early in the growth of the bacteria to ensure that it is thoroughly chilled before use. 3. If the cells are to be stored at -70C, use ESB buffer rather than TFB. ( ...)

. 59 60

Electroporation of E. coli

Introduction

Electroporation, originally developed as a method to introduce DNA into eukary-otic cells (7), has subsequently been extensively used for bacterial transformation (2,3). This procedure is an effective method for the transfer of DNA to a wide range of Gram-negative bacteria, such as Escherichia coli, and reports indicate that 109 electro-transformants per microgram of DNA can be achieved in this species (4,5). ( ...)

. 61 62

Materials

1. E. coli strain (see Note 2). 2. Luria-Bertani (LB) broth medium: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride. Adjust to pH 7.0 by addition of 5 N NaOH; autoclave. ( ...)

. 63

Methods

Preparation of E. coli Electrocompetent Cells 1. Streak a suitable E. coli strain onto an LB agar plate for single colonies and incubate at 37C overnight. 2. Inoculate 50 mL of LB medium with a single colony of freshly grown E. ( ...)

. 64 65

Notes

1. To electroportate ElectroMAXDH5a-E E. coli cells using the Bio-Rad Gene Pulser unit, the following conditions are used to yield approx 1.0 x 1010 transformants per microgram pUC plasmid DNA: 1.8 ( ...)

. 66 67

DNA Transfer by Bacterial Conjugation

1. Introduction

Bacterial conjugation is defined as contact-dependent transmission of genetic information from a donor bacterium to a recipient cell (7). Transfer of DNA by conjugation is often termed lateral or horizontal gene transfer, as opposed to vertical transfer by which genetic information is transferred from mother to daughter cells. ( ...)

. 68 69 70

2. Materials

1. Luria-Bertani (LB) broth medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride. Adjust to pH 7.0 by addition of 5 N NaOH and autoclave. 2. Antibiotics for selection of transconjugants. ( ...)

. 71

3. Method

1. Dilute overnight cultures of the donor and recipient strains 1 in 50 in fresh LB broth. Incubate at 37C with vigorous shaking until an OD600 of 0.6 - 0.8 is reached. 2. Mix different ratios of the donor and recipient strains in a sterile universal. ( ...)

. 72

4. Notes

1. Different ratios of donor to recipient strains are used to optimize the conjugation procedure. It may also be necessary to increase the cell biomass of the bacterial cultures. Optimization is particularly important if the recipient strain is not E. ( ...)

. 73

Cosmid Packaging and Infection of E. coli

Introduction

Cosmids are cloning vectors that were developed to enable large fragments of DNA to be cloned and maintained (1-3). Cosmid vectors allow the cloning of fragments up to 45 kilobases (kb) and are commonly used in genomic library construction. ( ...)

. 74 75

Materials

Ligation Reaction 1. Prepared (restriction digested and phosphatase treated) vector DNA (e.g., SuperCos I [Stratagene]). Store at -20C. 2. Prepared (restriction digested and phosphatase treated) genomic DNA. ( ...)

. 76

Methods

Ligation Reaction (1,3,6,10-13) 1. Set up the following ligation reaction in a microcentrifuge tube: 1.5-3.0 prepared genomic DNA (32-45 kb in length) (see Note 4). 1.0-3.0 prepared vector DNA. ( ...)

. 77 78

Notes

1. One of the most important things to consider when constructing a cosmid library is the efficiency of the packaging extracts. It is extremely important that the packaging extracts are not allowed to thaw before use. ( ...)

. 79 80

Isolation of Plasmids from E. coli by Alkaline Lysis

Introduction

Purification of plasmid DNA from Escherichia coli using alkaline lysis (1,2) is based on the differential denaturation of chromosomal and plasmid DNA in order to separate the two. Bacteria are lysed with a solution containing sodium dodecyl sulfate (SDS) and sodium hydroxide. ( ...)

. 81

Materials

Growth of E. coli 1. Luria-Bertani (LB) medium: 5 g/L yeast extract, 5 g/L NaCl, 10 g/L tryptone. 2. Appropriate antibiotics. Plasmid Isolation 1. STE (sucrose/Tris/EDTA) solution: 8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0 ( ...)

. 82

Methods

Growth of E. coli 1. Inoculate 3 mL of sterile LB medium containing the appropriate antibiotic with a single bacterial colony. 2. Grow with shaking at 37C overnight. Plasmid Isolation 1. ( ...)

. 83 84

Notes

1. The original protocol asks for 0.2 N NaOH. However, if the isolated plasmid DNA is to be used in sequencing reactions, reducing the NaOH concentration to 0.1 N is recommended. This reduces the amount of nicked and denatured DNA (see Note 7) without a significant impact on DNA yield. ( ...)

. 85 86

Isolation of Plasmids from E. coli by Boiling Lysis

Introduction

The boiling lysis procedure (1) is quick to perform and, therefore, especially suitable for screening large numbers of small-volume Escherichia coli cultures. It is described with different adaptations in a variety of protocol books (2,3). ( ...)

. 87

Materials

Growth of E. coli 1. Luria-Bertani (LB) medium: 5 g/L yeast extract, 5 g/L NaCl, 10 g/L tryptone. Autoclaved. 2. Appropriate antibiotics. Plasmid Isolation 1. STE solution: 8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0 ( ...)

. 88

Methods

Growth of E. coli 1. Inoculate 3 mL of sterile LB medium containing the appropriate antibiotic with a single bacterial colony. 2. Grow with shaking at 37C overnight. Plasmid Isolation 1. ( ...)

. 89 90 91

High-Purity Plasmid Isolation Using Silica Oxide

Introduction

The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. ( ...)

. 92 93

Materials

1. Silica oxide (Sigma): Dissolve in 250 mL of water, at 50 mg/mL, for 30 min. Remove the fines by suction and reconstitute the original volume. Add 150 of 37% HCl and autoclave. 2. P1: 50 mM Tris-HCl, 10 mM EDTA (pH 8.0 ( ...)

. 94

Methods

1. Harvest 1.5-2 mL of overnight cultures of Escherichia coli clones of interest in Eppendorf tubes by centrifugation at 1000g for 5 min and completely remove the supernatant (see Notes 2-4). 2. ( ...)

. 95

Notes

1. To facilitate pipetting, acetone should be stored at -20C. 2. This method can be scaled up for larger cultures; recommended volumes of solutions to use in the different formats are given in Table 1. ( ...)

. 96

High-Throughput Plasmid Extraction Using Microtiter Plates

1. Introduction

Plasmid extraction is typically performed to produce template DNA for a desired molecular biological reaction, or set of reactions, such as restriction endonuclease digestion (see Part 20), DNA sequencing (see Part 22), in vitro mutagenesis (see Parts 23-26), transformation (see Parts 4 and 5), transfection, or probe generation. ( ...)

. 97 98 99 100

Materials

96-Well Miniprep of Plasmid DNA 1. Circlegrow (Anachem, UK). 2. Ampicillin, or other antibiotic as appropriate. 3. Deep-well 96-well plates (Beckman). 4. Plate sealer (Costar, Corning). ( ...)

. 101

Methods

Plasmid Preparation in 96-Well Format 1. Fill each well of a 96-well-deep well plate with 1 mL of Circlegrow containing the appropriate antibiotic (typically ampicillin at a final concentration of 100 ^g/mL) (see Notes 1, 3, and 4). ( ...)

. 102

96-Well Miniprep of PAC, BAC, or Cosmid DNA

1. Fill each well of a 96-well deep-well plate (see Notes 1, 3, and 4) with 1.25 mL of 2X TY containing the appropriate antibiotic (typically 25 yg/mL of kanamycin for PACs and cosmids or 12.5 yg/mL of chloramphenicol for BACs). ( ...)

. 103

96-Well Single-Stranded DNA Prep from M13 Bacteriophage

1. Fill each well of a 96-well deep-well plate (see Notes 1, 3, and 4) with 1.25 mL of 2X TY that has been seeded with a 1% (v/v) inoculum of an overnight culture of an appropriate M13 host strain. ( ...)

. 104

4. Notes

1. Deep-well plates can be filled manually using a reservoir-based repeat pipettor such as an Eppendorf multipipet or by using a 1-mL-capacity multichannel pipet. For filling large numbers of boxes, a 96-well dispensing unit such as a Q-fill (25) is recommended. ( ...)

. 105 106

Isolation of Cosmid and BAC DNA from E. coli Daniel Sinnett and Alexandre Montpetit

Introduction

Cosmid and bacterial artificial chromosome (BAC) systems have been developed for the cloning of large DNA inserts averaging 40 kb and 130 kb (range: 90-300 kb), respectively. The resulting clones are more stable than yeast artificial chromosomes (YACs) and rarely chimeric, which makes them excellent tools for the generation of contiguous physical maps. ( ...)

. 107 108

Materials

1. Luria-Bertani (LB) medium: 10 g Bacto tryptone, 5 g Bacto yeast extract, 10 g NaCl; make up to 1 L with double-distilled water (ddH2O). Sterilize by autoclaving. 2. Terrific Broth (TB): 12 g Bacto tryptone, 24 g Bacto yeast extract, 10 mL of 40% (v/v) sterile glycerol, 17 mL of 1 M KH2PO4, 72 mL of 1 M K2HPO4; ( ...)

. 109

Method

(see Notes 1 and 2) 1. Prepare a 100- to 150-mL culture of the cosmid or BAC clone to be purified in LB or TB media containing the appropriate antibiotic (50 yg/mL kanamycin or 100 yg/mL ampicillin for cosmids and 12.5 ( ...)

. 110 111

Notes

1. This protocol can be adapted for minipreparation of DNA (3). 2. Cosmids and BACs can also be prepared using the CONCERT High Purity Plasmid Purification System from Invitrogen. However, it is necessary to modify their midiprep protocol as follows. ( ...)

. 112

Preparation of Single-Stranded DNA from Phagemid Vectors

Introduction

Single-stranded DNA (ssDNA) is the optimal template for most polymerase-based molecular-biology applications, including DNA sequencing and site-directed mutagenesis. Phagemids are chimeric vectors, derived from the ssDNA bacteriophages M13, fd, or f1, that normally replicate as plasmids in bacterial hosts (1) (see Part 2). ( ...)

. 113

Materials

Determination of Helper Bacteriophage Titer 1. Luria-Bertani (LB) broth: 5 g tryptone, 10 g yeast extract, 5 g NaCl. Bring to 1 L with water and autoclave. 2. LB agar: Add 15 g Bacto agar to 1 L of LB broth and autoclave. ( ...)

. 114

Methods

Determination of Helper Bacteriophage Titer 1. Prepare fifteen 5-mL aliquots of semisolid LB top agar in glass tubes and place at 42C to keep the agar molten. 2. Add 108 colony-forming units of a susceptible bacterial strain to 0.5 ( ...)

. 115 116

Notes

1. It is important to use good bacteriological techniques. Start with single-colony inoculae and carefully monitor the growth of the bacterial cultures. 2. The titer of the helper phage is important and should be determined in plaque assays as described in Subheading 3.1 ( ...)

. 117

Using Desktop Cloning Software to Plan, Track, and Evaluate Cloning Projects

Introduction

Manipulation and analysis of DNA sequences is often a complex task involving many steps, each of which must be carefully planned and executed. To facilitate this process, the number of steps should be minimized and each step analyzed to ensure that it has been completed successfully. ( ...)

. 118 119

Methods

Background As an example, an actual cloning project will be described. In this project, the coding sequence of a Drosophila heat-shock gene (hsp26) was cloned into a vector downstream of a regulated promoter. ( ...)

. 120

Annotating a Construct

Identifying Open Reading Frames 1. To characterize pRmHa3, coding regions were identified by highlighting open reading frames (ORFs) using Construct > Features > Find Open Reading Frames (see Fig. ( ...)

. 121 122

Making and Tracking New Constructs

Creating a New Generation 1. The source of the hsp26 coding sequence was the plasmid pJBl (see Fig. 5A). In order to isolate the segment containing the hsp26 coding sequence, pJ1B was digested with EcoRI and BamHI and the smaller of the two fragments generated was isolated using gel electrophoresis and gel extraction. ( ...)

. 123 124 125

3.5. Importing Sequences from Genbank

1. GCK's Deluxe Importing feature (File > Deluxe Import > Search GenBank) allows for a straightforward importing of GenBank (or EMBL) sequence files directly from the corresponding websites. ( ...)

. 126

Notes

1. Another useful feature of GCK, although not shown here, is the ability to create partial digests. For example, it is possible to specify that during a digest only five of six sites are cut. The resulting digest pattern will show complete digest fragments as solid black lines (bands) and partial digest fragments as dotted blue lines. ( ...)

. 127

Cloning in Plasmid Vectors

Introduction

A fundamental step in molecular biology is the cloning of a DNA fragment insert into a plasmid vector. This allows the cloned fragment to be replicated upon transformation of the recombinant molecule into a bacterial cell (see Parts 4 and 5) so that the DNA of interest can be investigated further. ( ...)

. 128

Restriction Digestion

The first step in cloning a DNA insert into a plasmid vector is cutting both vector and insert DNA with the appropriate restriction enzyme(s) to generate compatible ends. This may be a simple single digestion or a double digestion with two enzymes in the case of directional cloning. ( ...)

. 129

Introducing a Restriction Site

If the insert DNA does not contain convenient restriction sites, it is possible to generate a site at the desired position by amplifying the insert using the PCR primers designed with the restriction site. ( ...)

. 130

Converting an Overhang to a Blunt End

In cloning experiments where compatible ends are not available, it may be necessary to convert a 5' or 3' overhang to a blunt end (see Fig. 1B). Both bacteriophage T4 DNA polymerase and Escherichia coli DNA polymerase I large (Klenow) fragment have 5'3' polymerase activity and can be used to fill in 5' overhangs. ( ...)

. 131

Dephosphorylation of Vector DNA

Alkaline phosphatases are commonly used in cloning experiments to dephosphory-late the 5' ends of vector DNA. This prevents self-ligation of the vector, as the enzyme used to ligate the DNA molecules requires a 5'-phosphate group on one of the DNA substrates (see Fig. ( ...)

. 132

Phosphorylation of Insert DNA

Phosphorylation of insert DNA that lacks terminal 5' phosphates, such as PCR products and fragments with synthetic linkers, may be required in preparation for ligation. If the product is to be cloned into a nonphosphorylated vector, it is vital that phosphate groups are added to the insert. ( ...)

. 133

Ligation of Vector and Insert

The final step in cloning is the joining of the linear DNA fragments together, referred to as ligation. This involves creating a phosphodiester bond between the 3'-hydroxyl group of one DNA fragment and the 5'-phosphate group of another and is equivalent to repairing nicks in a duplex strand. ( ...)

. 134

Materials

Restriction Digestion 1. Appropriate restriction enzyme supplied with buffer; store at -20C. 2. 1 mg/mL bovine serum albumin (BSA), acetylated. 3. 0.5 M EDTA (pH 8.0). 4. ( ...)

. 135

Methods

Restriction Digestion Complete Digests 1. Add the following to a 1.5-mL Eppendorf tube on ice (see Note 3): DNA 0.1-1 yg (see Note 4) x yL Restriction enzyme (see Notes 5-7)1 yL Restriction enzyme 10X reaction buffer (see Note 8)2 yL BSA 1 mg/mL 2 yL Sterile double-distilled water to a final volume of 20 yL(see Note 9) 2. ( ...)

. 136 137 138 139

Notes

1. ATP should be present at a concentration of at least 1 yM. 2. The buffer is usually provided or prepared by the manufacturer as a 10X concentrate, which, on dilution, yields an ATP concentration of approx 0.2 ( ...)

. 140 141 142 143 144 145

Extraction of DNA from Agarose Gels

1. Introduction

A common step in cloning experiments is the purification of DNA fragments prior to ligation. Often, both the insert and vector DNA fragments will be derived from restriction endonuclease digests and, thus, will be mixed with enzymes, salts, and possibly other DNA fragments that may inhibit the ensuing ligation reaction. ( ...)

. 146

2. Materials

2.1. Low-Melt Agarose Protocol 1. Low-melting-point agarose. 2. 5X TBE buffer: 54 g Tris base, 27.5 g boric acid, 20 mL of 0.5 M EDTA (pH 8); make up to 1 L with distilled water. Dilute the stock to give a 1X working solution immediately prior to use. ( ...)

. 147

3. Methods

3.1. Low-Melt Agarose Protocol This protocol makes use of low-melt agarose. In its simplest form, this protocol is more an avoidance of extraction rather than an extraction per se. It can be modified to increase the purity of the DNA sample (see Subheading 3.1 ( ...)

. 148 149

4. Notes

1. If TBE electrophoresis buffer is inhibitory to the downstream application and the DNA is to be used without further purification, it is possible to use a different buffer system for gel electrophoresis. ( ...)

. 150

Cloning PCR Products with T-Vectors

1. Introduction

1.1. Overview of PCR Since it was described in 1988 (/), the polymerase chain reaction (PCR) has been a valuable tool for molecular biologists. PCR allows researchers to produce a large quantity of a desired DNA fragment while requiring only a small amount of template. ( ...)

. 151 152 153 154 155

2. Materials

2.1. Preparation of an Xcml-Based T-vector 2.1.1. Construction of Custom T-Vector 1. Sterile distilled water. 2. 1 yg/yL Oligonucleotide #1 in water: 5'-GATCCAAGCTTCCCATGGCGCCATGTCAT GAGTGGCTGCA-3'. ( ...)

. 156

3. Methods

3.1. Preparation of an Xcml-Based T-Vector 3.1.1. Construction of Custom T-Vector 3.1.1.1. Preparation of Oligonucleotides 1. Combine 25 yL oligonucleotide #1 and 25 yL oligonucleotide #2 in a microcentrifuge tube. ( ...)

. 157 158

4. Notes

1. In the past, researchers have had difficulty with XcmI performance and this hampered preparation of T-vectors. However, higher-quality enzyme is now available and this problem is not as prevalent as it once was. ( ...)

. 159 160

Construction of Genomic Libraries in Vectors

1. Introduction

Lambda (k) bacteriophages are viruses that specifically infect bacteria. The genome of k-phage is a double-stranded DNA molecule approx 50 kb in length (7). In bacterial cells, k-phage employs one of two pathways of replication: lytic or lysogenic. ( ...)

. 161 162

2. Materials

2.1. Preparation of Genomic DNA for Cloning 2.1.1. Purification of Genomic DNA We suggest the use of the Wizard Genomic DNA Purification Kit (Promega); items 1-3 are components of that kit. ( ...)

. 163 164

3. Methods

3.1. Preparation of Genomic DNA for Cloning 3.1.1. Purification of Genomic DNA 1. Mince 150 mg of tissue in 40 yL/mg of ice-cold nuclei lysis buffer. Homogenize on ice using 10-15 strokes with a Teflon pestle. ( ...)

. 165 166 167 168 169 170 171

4. Notes

1. A number of similar ready-to-use k-vectors are commercially available for genomic library construction. For example, LambdaGEM-11 BamHI arm is a similar vector and is currently available (Promega). ( ...)

. 172 173

Rapid Screening of Recombinant Plasmids

1. Introduction

Construction of recombinant plasmid DNA is one of the cornerstones of molecular biology. The ability to clone DNA in a plasmid vector opens doors to downstream applications such as amplification of DNA, expression of desired genes, and construction of DNA libraries. ( ...)

. 174 175 176

2. Materials

2.1. Blue-White Selection 1. Luria-Bertani (LB) agar plates: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar. Add water to 1 L and autoclave to sterilize. Cool to approx 50C, add antibiotics as appropriate, and pour approx 20 mL into each Petri dish. ( ...)

. 177

Methods

3.1. Blue-White Selection 1. Transform E. coli with the ligation reaction using methods described in Parts 4 and 5. 2. Prepare plates by adding 40 yL of 20 mg/mL X-gal and 50 yL of 0.1 ( ...)

. 178 179

Notes

1. Instead of spreading IPTG and X-gal on to LB agar plates, they can be added to the agar mixture before plates are poured (final concentration: 6 mM IPTG and 0.3 mg/mL X-galin LB agar). However, this approach uses more IPTG and X-gal and these plates have a fairly short shelf life. ( ...)

. 180

Restriction Analysis of Recombinant Plasmids

Introduction

A key step in the construction of recombinant plasmids is the verification of the successful cloning of insert DNA into the vector. A number of commonly used plas-mids facilitate phenotypic selection and/or screening methods for rapid identification of insert-containing clones. ( ...)

. 181

Determination of Appropriate Restriction Enzymes and Digests

The choice of appropriate enzyme(s) for the restriction analysis of the clone will depend on the plasmid and insert involved. Several criteria may influence this decision. For example, the resulting DNA fragments need to be within a size range detectable on a gel (see Note 1) and the fragments of interest must be easily distinguishable from each other. ( ...)

. 182

Materials

2.1. Restriction Enzyme Digestion 1. Predicted restriction map of the plasmid clone. Prepare a map for a clone with the insert in each of the two possible orientations if applicable. ( ...)

. 183 184

Methods

3.1. Restriction Enzyme Digestion 1. Thaw all solutions, except the enzyme, and keep on ice. 2. Using a final volume for the digest of 20 yL (or up to 50 yL if the DNA is dilute), add the following into a sterile Eppendorf tube: 1/10 volume reaction buffer (see Note 9). ( ...)

. 185

Notes

1. A DNA fragment of a size between 100 and 10,000 base pairs is ideal. DNA fragments that are larger or smaller than this will migrate in the gel, but may blur or fail to resolve (1,4,5). If trying to resolve and analyze fragments larger than 5 kb, a gel that is longer than a mini-gel is required. ( ...)

. 186 187 188

Screening Recombinant DNA Libraries

Introduction

A recombinant DNA library typically represents part or all of an organism's genomic DNA or mRNA (represented as cDNA) cloned into vectors and stored as a collection of thousands of transformants. ( ...)

. 189

Screening Methods

1.1.1. Phenotypic Screening In a small number of cases, a cloned fragment of DNA will possess an intact gene that encodes a protein of discernable function. Some examples are genes encoding pigments, secreted enzymes, or assayable metabolic functions. ( ...)

. 190 191 192 193 194

Materials

2.1. Preparing the Membrane 1. Recombinant library stored in multiwell plates. 2. LB agar: 10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, and 15 g/L bacto-agar. Sterilize by autoclaving. ( ...)

. 195

Methods

3.1. Preparing the Membrane 1. Remove a multiwell plate containing the library from the freezer and allow the bacterial suspensions to thaw on ice. 2. Label the edge of a nitrocellulose membrane with the specific information about the library (plate number, date, etc.) ( ...)

. 196 197 198 199 200 201

Sequencing Using Fluorescent-Labeled Nucleotides

1. Introduction

The most widespread method used for DNA sequencing today is the Sanger dideoxy method that was first described in 1977 (7). This method takes advantage of the requirement for a free 3' hydroxyl group to form the necessary phosphodiester bridge between two nucleotides during DNA polymerization. ( ...)

. 202 203

2. Materials

2.1. Sequencing Reaction Setup 1. Purified plasmid (100-500ng/yL), polymerase chain reaction (PCR) product (10 ng per 100 bp), or bacterial artificial chromosome (BAC) clone (200-600 ng/yL) (see Note 1). ( ...)

. 204 205

3. Methods

3.1. Sequencing Reaction Setup (see Note 8) 3.1.1. Reaction Setup for Slab Gel Sequencing of Plasmids and PCR Products 1. Add 1-5 yL of plasmid DNA or 10 ng per 100 bp PCR template DNA to a sterile, thin-wall PCR tube or a 96-well PCR plate. ( ...)

. 206 207 208 209

4. Notes

1. Template DNA must be of extremely high quality. When using the ABI dye chemistries, purify plasmid templates using Qiagen mini-spin kits (www.qiagen.com) or Promega Wizard preps (www.promega.c ( ...)

. 210 211 212

Site-Directed Mutagenesis Using the Megaprimer Method

1. Introduction

Site-directed mutagenesis (SDM) is used to introduce a defined mutation into target DNA of known sequence to study, for example, gene expression or protein structure-function relationship. A number of polymerase chain reaction (PCR)-based mutagenesis methods have been developed (7). ( ...)

. 213 214

Materials

1. DNA template and plasmid carrying the gene sequence to be mutated. 2. Oligonucleotide primers: Two external primers (forward and reverse) and one internal mutagenic primer. 3. 5 U/yL Pfu DNA polymerase and 10X reaction buffer (Stratagene, La Jolla, CA). ( ...)

. 215

Methods

3.1. Primer Design 3.1.1. Mutagenic Primer We design primers that are 22-24 bp in length. This gives sufficient length for incorporating the required base-pair change and to give the desired annealing temperature (Tm) > ( ...)

. 216 217

4. Notes

1. Template DNA should be kept at a low concentration (e.g., around 1 ng). Excess template leads to high levels of wild-type sequence being carried over into the second-round PCR, which results in a high level of wild-type sequence in the second-round PCR products. ( ...)

. 218

Site-Directed Mutagenesis by Inverse PCR

1. Introduction

Site-directed mutagenesis has revolutionized the study of protein structure and function by enabling the controlled and systematic production of mutant proteins. Early methods of site-directed mutagenesis involved the use of a mutated oligonucleotide primer to prime synthesis of a target single-stranded DNA template. ( ...)

. 219 220

1.1. Enzymatic Inverse PCR Using Type IIS Restriction Endonucleases

Enzymatic inverse PCR using Type IIS restriction endonucleases (EIPCR-IIS) is a significant improvement over the classical method (4). In this technique, the 5' termini of both primers contain a unique Type IIS restriction site, such as SapI. ( ...)

. 221

1.2. Enzymatic Inverse PCR Using Type II Restriction Endonucleases

The author's laboratory has adapted the original EIPCR protocol for the use of class II restriction enzymes (5), thereby extending the versatility of the technique. The principle difference from EIPCR-IIS is that in stage 1 of this process, a unique Type II enzyme recognition site is artificially introduced into the construct. ( ...)

. 222 223 224

3. Methods

3.1. EIPCR-IIS The EIPCR-IIS protocol is outlined in Fig. 2. 3.1.1. Primer Design Careful primer design is crucial for the success of any DNA amplification experiment and is particularly critical when designing primers for site-specific mutagen-esis. ( ...)

. 225 226 227 228

4. Notes

1. Magnesium chloride is required for the activity of the DNA polymerase and is typically used at 1.5 mM final concentration, although variation of Mg2+ levels between 1.0 and 2.5 mM MgCl2 can increase the specificity of the amplification reaction (7). ( ...)

. 229 230 231 232

Creating Nested DNA Deletions Using Exonuclease III

1. Introduction

DNA fragments cloned into plasmids are frequently greater than 500 base pairs in length and thus may be too long to sequence from a single primer-binding site in the vector. An efficient way to sequence such large DNA inserts is to generate a nested set of deletions in the target DNA, effectively moving the priming site closer to the sequence of interest. ( ...)

. 233 234

2. Materials

2.1. Restriction Enzyme Digestion 1. CsCl/ethidium bromide-purified plasmid DNA (see Note 1). 2. Restriction enzymes and corresponding 10X buffers suitable for generating a 3' recessed terminus or blunt end, and a four nucleotide 3' overhang (see Notes 2-4). ( ...)

. 235

3. Methods

3.1. Restriction Enzyme Digestion 1. Digest 10 yg of the plasmid DNA with the restriction enzyme that generates the 3' recessed terminus or blunt end according to the manufacturer's instructions (this enzyme site must lie closest to the target DNA). ( ...)

. 236 237

4. Notes

1. The generation of ordered sets of deletions by this method relies on the uniform digestion rate of exonuclease III. However, the enzyme also digests from nicks in double-stranded DNA molecules, creating single-stranded gaps. ( ...)

. 238 239

Transposon and Transposome Mutagenesis of Plasmids, Cosmids, and BACs

1. Introduction

Transposons are mobile genetic elements with the capacity to "jump" to new target DNA. Although first discovered in Zea mays by McClintock (7), they are present in DNA genomes of species from all kingdoms. ( ...)

. 240 241 242 243 244

2. Materials

2.1. Tn7-Based In Vitro Mutagenesis of Plasmids and Cosmids 1. Tn7 transprimer kit: Genome Priming System, GPS-1 (New England Biolabs). This kit contains the basic transprimer plasmid (with either chloramphenicol- or kanamycin-resistance markers), purified Tn proteins (A, B, C*), Tn reaction buffers, and Tn7-specific sequencing primers. ( ...)

. 245

Methods

3.1. Tn7-Based In Vitro Mutagenesis of Plasmids and Cosmids 1. Thaw the contents of the Tn7 GPS transprimer kit and place on wet ice. 2. Prepare the following reaction mixture in a 1.5 ( ...)

. 246 247 248 249

In Vitro Transcription and Translation

1. Introduction

In this part, we describe the use of plasmid vectors in transcription and translation systems in vitro to investigate aspects of the biology of the gene and the protein for which it codes. An in vitro, or cell-free, assay reproduces a relatively complex physiological process by mixing the essential purified components of the system under controlled conditions outside of the cell. ( ...)

. 250 251 252 253 254 255

2. Materials

2.1. In Vitro Transcription from Phage Promoters (see Note 1) 1. Linear template DNA (0.2-1 yg/yL) (see Notes 2-4). 2. 5X transcription buffer: 200 mM Tris-HCl (pH 7.9), 30 mM MgCl2, 10 mM spermidine, 50 mM NaCl. ( ...)

. 256

3. Methods

3.1. In Vitro Transcription from Phage Promoters 1. Prepare the reaction mixture at room temperature (see Note 8), as follows: 5X Transcription buffer 4 yL 100 mM DTT 2 yL Ribonuclease inhibitor 20-40 U 2.5 ( ...)

. 257

Notes

1. All reagents, except items 1, 8, and 11, can be purchased either as separate items or as a Riboprobe kit from Promega. Store at -20C. 2. The gene of interest must be cloned under the control of a strong promoter such as T3, T7, SP6, or an appropriate E. ( ...)

. 258 259

Vectors for the Expression of Recombinant Proteins in E. coli

1. Introduction

Escherichia coli is the most commonly used and best characterized organism for overexpressing foreign and nonforeign proteins. The use of E. coli confers several immediate advantages to the user: rapid and high-level expression as a result of the speed of cell growth to high density; ( ...)

. 260

2. Cloning Into Expression Vectors

2.1. Transcription Versus Translation Vectors There are two types of expression vector: transcription vectors and translation vectors. Transcription vectors are utilized when the DNA to be cloned has an ATG start codon and a prokaryotic ribosome-binding site. ( ...)

. 261 262

3. Expression System

3.1. Promoters Proper promoter selection is of the utmost importance when designing an expression system. In fact, expression vectors were originally classified by the nature of their promoters because a strong promoter was considered the most important asset of these vectors (9). ( ...)

. 263 264 265 266 267

4. Gene Fusions

4.1. Subcellular Localization The localization of a protein in the host cell may affect its production and tertiary structure. Recombinant proteins can be directed to one of three compartments: cytoplasm, periplasm, or extracellular medium (secreted). ( ...)

. 268 269 270

5. Troubleshooting Tips for Insoluble Proteins

Certain vectors and host strains enhance the likelihood of expressing a soluble protein. One approach to increasing the soluble yields of aggregated proteins is to improve folding of newly synthesized proteins through the co-overexpression of cyto-plasmic molecular chaperones (70). ( ...)

. 271

6. Conclusions

The E. coli plasmid vectors available to researchers are continually fine-tuned, making it easier to express a wide variety of proteins in any given expression system. A list of commercially available expression vectors is included in Table 1. ( ...)

. 272

Expression of Recombinant Proteins From lac Promoters

1. Introduction

The Gram-negative bacterium Escherichia coli enjoys widespread use in modern biology as both a model organism and a microbiological tool. One of the keys to its popularity lies in the functionality of the lac operon. ( ...)

. 273 274 275 276 277 278

2. Materials

2.1. Expression of Recombinant Protein 1. Plasmid-bearing recombinant gene of interest (see Note 1). 2. E. coli host strain (see Subheading 1.3.). 3. LB growth medium, prepared according to manufacturer's instructions (see Notes 2 and 3). ( ...)

. 279

3. Methods

3.1. Expression of Recombinant Protein 1. Grow a 5-mL overnight culture of the expression construct at 37C with vigorous shaking. Use the same growth medium as will be used for expression. ( ...)

. 280 281 282 283

Plasmid-Based Reporter Genes

1. Introduction

Reporter genes encode easily measurable traits. Most commonly, they are used to investigate the expression of other genes for which functional assays are not available or for which measurement of expressed product is difficult. ( ...)

. 284

1.1. Choosing a Reporter Gene Vector

Many of the reporter genes listed in Table 1 are available in different types of vectors that have been tailored to specific applications. In choosing a vector system, considerations should be given to several factors. ( ...)

. 285 286

Materials

2.1. p-Galactosidase Assay 1. PM2 buffer: 36 mM NaH2PO4, 67 mM Na2HPO4, 0.1 mM MgCl2, 2 mM MgSO4 (see Note 1). 2. PM2SH: Add 135 yL of p-mercaptoethanol to 50 mL of PM2 buffer (see Note 2). ( ...)

. 287

Methods

3.1. p-Galactosidase Assay 1. Grow cultures of the strains to be assayed. The assay should be performed using mid-log cultures (see Note 4). 2. Chill the cultures on ice for at least 10 min, to prevent further growth, and measure the cell density at OD600. ( ...)

. 288

Notes

1. These solutions can be autoclaved or filter sterilized. Their performances are not affected by storage at ambient temperature for up to 6 mo. 2. Prepared fresh as needed. 3. Keep cold at approx 4C. ( ...)

. 289 290

Plasmid-Based Reporter Genes

Introduction

Green fluorescent protein (GFP) of the jellyfish Aqueorea victoria is a 238-amino-acid, 28-kDa protein that absorbs light with an excitation maximum of 395 nm and fluoresces with an emission maximum of 509 nm (7). ( ...)

. 291 292 293

Materials

2.1. Direct Colony Examination Short-wavelength UV lamp or appropriate imaging device. 2.2. Fluorescence Microscopy 1. Dulbecco's phosphate-buffered saline (DPBS, pH 7.4). 2. ( ...)

. 294

Methods

3.1. Colony Examination This is the quickest and easiest way of visualizing fluorescent bacterial colonies. In the case of questionable fluorescence, use the fluorescence microscopy protocol in Subheading 3.2 ( ...)

. 295 296 297

4. Notes

1. Grow cells in the appropriate liquid medium to exponential phase: l x 108 to l x 109 cells/ mL for bacteria, l x 106 to l x 107 for yeast, and l x 104 and l x 105 for mammalian. 2. Mix 5 yL of liquid culture with 5 yL of molten l% low-melting-point agarose at 37C. ( ...)

. 298 299

.