Project Overview

You have just begun your first job as a technician in a research lab. Your boss has given you the job of subcloning a gene she wishes to work with, from an old style vector into pUC18. You have been given two strains of E. coli; one carries the source plasmid for GeneA, and the other carries the new vector pUC18. You have found two different approaches to subcloning; one is commonly referred to as shotgun cloning and the other can be called targeted cloning. You have decided to try both methods and compare the results, so that for future cloning projects you can choose the most appropriate method.

Your have a book of protocols, but you need to plan a strategy, and use the appropriate protocols to achieve your goal. The basic steps involved in producing recombinant DNA are outlined in Figure 1.

General DNA cloning steps

  1. Plan the cloning strategy. This includes selecting a method for obtaining the DNA fragment to be cloned, selecting a vector into which the DNA fragment will be incorporated, and selecting the bacterial host that will be used to produce sufficient quantities of the cloned DNA fragment for further characterization.
  2. Isolate the vector and the DNA fragment to be cloned. For the vector this involves growing an appropriate bacterial strain carrying the vector, and isolating the vector DNA away from other cellular components. The method used to obtain the DNA for cloning depends on where it currently resides (in a plasmid, in genomic DNA, in mRNA etc.).
  3. Digest or otherwise break up the DNA of interest, and digest the vector with appropriate restriction enzymes.
  4. Ligate the digested vector with the DNA fragment(s) of interest.
  5. Screen the new plasmids to find the desired recombinant plasmid(s). This involves transforming host bacteria with the ligation mixture and applying selection methods to isolate single bacteria carrying the newly created plasmids. Plasmids are then re-isolated and checked for correct structure.
Figure 1. Cloning Steps

Step I. Planning the cloning strategy

In order to plan a cloning strategy, you must first be familiar with the many available approaches and several molecular protocols. Since you are new to molecular biology the cloning strategy has already been laid out for you. However, by the end of the semester, you will be expected to be able to plan out your own hypothetical cloning strategy. The main things you need to plan out before you start are what restriction enzymes you will use, what vector you will work with, what bacterial strain will be used for the transformation and what screening strategy you will use determine which bacterial colony carries the recombinant plasmid you want.

Selecting the enzymes

A simple way to subclone from one plasmid to another is to use restriction enzymes. Knowing we have access to a plasmid carrying the piece of DNA we wish to clone, our first step is to look at the plasmid maps of both the source plasmid, pKC7, and the recipient plasmid, pUC18. Plasmid maps typically show the location of important genes, cis-acting elements, and recognition sites of enzymes that cut the plasmid in one or two places (many other enzymes may also cut the plasmid in three or more places, but generally these are not useful for cloning purposes).
The following criteria are used in selecting restriction sites for subcloning.

  • Enzymes must not cleave within the DNA segment to be cloned.
  • If possible, enzymes should cut the source plasmid only once.
  • Enzymes should cut relatively close to the margins of the DNA segment you wish to clone. Particularly, you don't want to include any functional sequences that could interfere with what you want to do with the new clone.
  • Enzymes must produce ends compatible with the vector (usually in the MCS).
  • Depending on the bacterial strain used to multiply the new plasmid, you may have to consider whether the site you have chosen is modified or restricted in the recipient strain.

Looking at the map of pKC7 you will note that recognition sites for Hind III and BamH I exist on either side of GeneA, but not within the gene. These sites also are found in the pUC18 MCS and therefore, are a good choice for the subcloning. Because we are using a different enzyme to cut each end of the insert, this is referred to as directional cloning.

Step II. Producing the starting material

The method used to producing starting material depends on what is available. The source plasmid, pKC7, and the vector, pUC18, are available carried in E. coli strain DH5α. Growing the strains in liquid media and isolating the plasmids produce large amounts of plasmid. The amount of culture needed depends on the approximate yield of plasmid DNA per milliliter of culture.

Plasmid yield per milliliter of stationary culture is primarily dependent on the plasmid copy number (the average number of plasmids per cell). Plasmid copy number is primarily controlled by the plasmid's replicon. A DNA replicon includes the DNA replication origin (the ori) and DNA encoded replication control elements. In plasmids, the ori and regulatory elements are usually found close together.

Over 30 different plasmid replicons have been described but almost all plasmids used in molecular cloning carry a replicon derived from pMB1 (Sambrook and Russell, 2001). The naturally occurring pMB1 replicon has a copy number of 15 to 20 plasmids per bacterial cell. However, vectors derived from pMB1, such as the pUC family, carry highly modified replicons that can maintain hundreds of copies per cell.
The plasmid pKC7 is a pBR322 derivative that carries the un-altered pMB1 replicon and under normal conditions, is maintained at 15 to 20 copies per cell. On the other hand, pUC18, is maintained at 500 to 700 copies per cell.

Isolating plasmid DNA

There are many different protocols for isolating plasmid DNA from bacterial cells but they all contain the same two basic steps: lyse the cells and separate the plasmid DNA from the other cell components.
To purify plasmid DNA, we will use a kit manufactured by Fermentas. This kit uses the tried and true method of alkaline lysis followed by neutralization to lyse the bacterial cells and separate the plasmid DNA from most other cell contents. The plasmid is further purified using a silica membrane. The ultra clean plasmid DNA produced using this kit will be compatible with all downstream cloning steps.

A molecular biology kit is a commercially prepared set of reagents for a specific protocol. Generally some of the reagents and/or the protocol is proprietary in nature and has been extensively developed and tested to make the protocol both simpler and more dependable than the ‘from scratch’ version. Think of a kit as a cake mix – except that kits usually produces a higher quality product than the homemade version.

Step III. Produce and prepare the DNA fragments for cloning

The next step is to digest the purified plasmids with the appropriate restriction enzymes; in our case these will be Hind III and BamH I. As with any enzyme, a restriction enzyme has specific requirements for pH, salt concentrations, temperature and cofactors in order to function correctly. Unfortunately, not all restriction enzymes have the exact same requirements. This means that when digesting with two enzymes, either the requirements for both enzymes must be met simultaneously or the two digests must be carried out sequentially. Fortunately conditions can usually be created that, while not ideal for both enzymes, will allow both enzymes to function well enough to get the job done in a single digest.

Once digestion is complete the fragments must be prepared for ligation. As mentioned in the project outline, we are going to use and compare, two different methods for recombining the plasmid fragments. In the shotgun cloning method the pieces from the two digested plasmids are simply mixed together. In the targeted cloning method we first purify the desired fragments away from the extraneous plasmid material and then mix these pieces together. In our case, the pKC7 fragment containing GeneA will be isolated using gel purification. This method involves separating the restriction fragments on a gel, cutting out the desired band and purifying the DNA from the gel slice. To gel purify the fragment we will use a kit produced by Qiagen. The pertinent information from the manufacturer's manual is in the protocol section.

Step IV. Ligation

In order to create a functional recombinant plasmid, we need to covalently join the restricted pieces together using the enzyme DNA ligase. As with all enzymes we use, ligase is naturally occurring. In vivo, DNA ligase is used during DNA synthesis to join DNA ends in lagging-strand synthesis of DNA. The DNA ligases commonly used in cloning originated from either bacteria or from bacteriophages.

DNA ligase catalyzes the formation of phosphodiester bonds between adjacent 3'–hydroxyl and 5'–phosphoryl termini of nucleic acids. Luckily this is exactly the kind of ends left by the restriction enzyme digestion of the DNA. Note that the 3' OH and 5' P must be close together. This is why overhangs must be complementary to each other, allowing base pairing which brings the ends of the molecules close together.

Step V. Screening the newly constructed plasmids

Ligation reactions often produce more than one product and screening is required to find the desired recombinant. Screening involves three main steps: transforming bacterial cells with the ligation products, selecting for bacteria carrying recombinant plasmid, and checking the plasmids to make sure they carry the correct insert.


Transformation refers to the process whereby cells uptake 'naked' foreign DNA. When cells are amenable to transformation they are referred to as competent. Occasionally, transformation occurs naturally, but most bacterial species need to be made competent by chemical or mechanical treatments. We will use the traditional calcium-chloride method to produce competent cells.

Screening transformants

Antibiotic selection

In any transformation experiment, only a subset of the bacteria will actually take up DNA. This means we need a way of quickly identifying which bacteria are transformed. All cloning vectors carry some type of selection marker. Often this marker is a gene encoding antibiotic resistance. Following transformation, the bacteria are plated on the appropriate selective medium (the antibiotic in the media must match the resistance gene encoded by the plasmid). Only bacteria that are transformed with the vector will be able to grow on this media. Our vector, pUC18, carries the gene for ampicillin resistance (β-lactamase).

Blue/white screening

Depending on the vector chosen, we may also be able to screen for bacteria that carry a recombinant vector (vector containing an insert in the MCS). In our case, visual screening is possible because the vector pUC18 carries a lacZ gene fragment. Bacteria carrying an insert in the pUC18 MCS will produce white colonies when grown on media containing X-gal. If no insert is present, the colonies will be blue.

To understand blue/white screening, you need to know a bit about β-galactosidase structure and function. β-galactosidase is naturally found in E. coli and is responsible for cleaving lactose into glucose and galactose, which can then be further metabolized by the bacterium. Functional β-galactosidase is a homo-tetramer, normally encoded by a single gene (the lacZ gene).

Researchers discovered that the single polypeptide can be divided into two pieces (by dividing the gene into two pieces) and if both polypeptide pieces are in the same cell, they can associate, forming a fully functional β-galactosidase enzyme. The two pieces are a short N terminal segment called the α-fragment and a much longer C-terminal fragment called the ω-fragment. The four polypeptides will form a tetramer only if the α-fragment is present. The ω-fragment contributes the enzyme activity. These findings were exciting because they suggested that proteins are made up of domains, with each domain contributing a distinct function to the protein.

For molecular biologists, understanding β-galactosidase function led to a new cloning tool. E. coli strains were created in which the lacZ gene was mutated by removing a small piece of DNA that encodes the first 41 amino acids. Bacteria with this mutation (lacZΔM15) cannot metabolize lactose. If the missing DNA sequence is supplied to the bacteria on a plasmid, the cells can once again metabolize lactose.

Plasmids that produce the α-fragment were constructed. A small DNA fragment carrying several restriction enzyme sites (the MCS) was inserted into the DNA encoding the α-fragment. The small numbers of amino acids encoded by the MCS do not inhibit enzyme activity, but when a piece of DNA is cloned into the MCS, enzyme activity is lost because the α-fragment is inactive (either because of a frame shift or nonsense mutation, or due to the size of the insert).

When E. coli cells with the lacZΔM15 mutation are transformed with a plasmid carrying an uninterrupted α-fragment, they will metabolize lactose (Figure 2). Bacteria transformed with a plasmid that carries an insert in the α-fragment will not metabolize lactose (Figure 3). Thus, based on ability to metabolize lactose, we can determine which E. coli cells are transformed with a vector carrying an insert.

Screening bacteria using metabolism abilities is cumbersome, as it requires replica plating all the transformants. Fortunately, β-galactosidase will cleave the chemical X-gal, producing a blue colour. Thus, when plated on media containing X-gal, E. coli harbouring a plasmid carrying an insert will form white colonies, while E. coli harbouring a plasmid without an insert will be blue.

Figure 2. LacZ complementation - no insert.
Figure 3. LacZ complementation - with an insert.

Identifying recombinant DNA

Finally, once you have narrowed down the colonies likely to carry the desired recombinant, the recombinant plasmids harboured by the colonies must be directly screened. Even when you expect only one recombinant type, it is important to confirm the correct construct is present, as visual screening methods are not 100% reliable. Often the recombinant plasmid will be used in many experiments, and you would hate to find out several experiments down the road that you had not constructed the desired clone.

There are several approaches to screening the recombinant plasmids; we will use restriction mapping. Because we know the maps of our starting plasmids, we can predict the sizes of fragments that will be produced when we digest the desired recombinant. It is simply a mater of choosing restriction enzyme(s) that will produce a diagnostic set of fragments (visualized using agarose gel electrophoresis).

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