Molecular Biology Laboratory Topics   

Cloning

Cloning Vectors

Cloning is to make more of identical items, in the case of DNA, to copy a specific nucleotide sequence by manipulating the replication system. Cloning vectors are usually plasmids or bacteriophages.

A plasmid, also know as extrachromosomal genetic element, is a small molecule of circular DNA outside a bacterial chromosome. Plasmids replicate independently from the chromosome and confer specific characteristics to the bacteria, like antibiotic resistance, or the ability to form a probuds (F1 plasmid) to inject DNA from a "male" to a "female" E. coli (conjugation).

Many different plasmids have been modified to be used in systematic cloning of specific DNA fragments. In order to clone our DNA sequence of interest using a plasmid we need to:

  1. Insert the sequence into the plasmid
  2. Replicate the recombinant plasmid
  3. Isolate plasmid DNA
  4. Separate our cloned sequence from the plasmid

Our DNA fragment of interest is inserted into the circular plasmid DNA, producing a hybrid plasmid that can be used to transfom bacteria. The recombinant plasmid is replicated in the transformed bacteria.

In order to prepare a plasmid to receive an insert, the plasmid should be cut with the samr restriction endonuclease as the insert was (see next section). To prevent the vector from reannealing onto itself instead of taking in the insert, its terminal phosphate groups may be removed with alkaline phosphatase, usually calf intestinal alkaline phosphatase (CIAP).

Ligase is used to ligate our open vector and DNA. After the ligation reaction is completed, spin dialysis or other method is used to remove leftover reagents aand get the purified recombinant plasmid.

Plasmids must be inserted into bacteria in order to replicate. The process of getting the plasmid DNA into the bacteria (usuallly E. coli) is known as transformation. Bacteria that have succesfully aquired our recombinant plasmid are said to be transformed. Some plasmids replicate more often than others, depending how strong is the signal of their replication origin. The copy number of a particular plasmid is the number it usually replicates to in a bacterial host.

Example Procedure: Plasmid DNA Isolation

This procedure is similar to the bacterial DNA isolation. Since the plasmids are usually contained inside bacteria, the plasmid DNA is separated from the genomic bacterial DNA by size. NaOH is used to denature DNA, i.e. make it single stranded. The DNA mixture is then allowed to renature. The chromosonmal DNA will not be able to renature because it is too big and proteins become attached to it. The plasmid DNA will renature after adding sodium acetate (NaOAC) to neutralyze the negative charges in DNA, allowing individual DNA molecules to move closer to each other. A first precipitate (?) will be a mixture of chromosome and proteins. DNA is insoluble in ethanol, thus is precipitated. A salting-out step will precipitate proteins and some polysacharides (step 11 p 4). By end of step 12, pellet has plasmid DNA, residual genomic DNA, RNA (add RNase in step 13), residual protein, carbohydrates (add cesium chloride), and lipids. EtBr binds to DNA and makes it lighter (because tBr has a ligther molecular weigth), binds more to chromosomal. Plasmid DNA may supercoil: some of the DNA in the chromosomal band may have relaxed plasmid; supercoiled plasmid separates into denser band. After removing the supercoiled plasmid band, it has DNA EtBr and CsCl (?). Butanol is added to remove the EtBr. CsCl is removed by dialysis. This procedure is simplified by using a Maxi Prep kit, which uses column chromatography. The DNA extracted by this traditional procedure can last forever, DNA extracted by Maxi Prep may last up to 6 months due to residual proteins.

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Example Procedure: Cloning Using a Plasmid Vector
Part 1: Preparation of Vector

Cloning procedures usually start by preparing both the vector and the piece of DNA we want to clone (the insert). The plasmid DNA is digested with the same restriction endonuclease used to cut the insert (see Part 2 below). A stop mix containing EDTA, SDS, glycerol and dye is used to stop the nuclease by chelating divalent metals needed for the reaction. The linear plasmid may be dephosphorilated to minimize the probability of reanneasling to itself. Use the proper buffer for the phosphatase, for example CIAP requires Zn2+ and a pH = 9.3. Extractions with phenol and chloroform remove any lipids, carbohidrates and proteins in the mixture.

  1. Incubate 2 µg or more of DNA with the enzyme and a buffer specific for that enzyme at the recomended temperature for at least 1hours or overnigth. An example reaction mixture follows:

    10 µl DNA
    2 µl buffer
    1 µl enzyme (10 U)

  2. To verify that the vector is open, run about 1 µl of the reaction mixture, mixed with an equal volume of 5x stop mix on a 1% agarose gel along with a molecular weight marker. If the vector is compleately cut, you should see a single band at the appropriate size for the linear vector. If there is any circular or supercoiled vector present, there will be bands at higher and lower sizes, respectively.

  3. If the plasmid was compleatelly linear, add 5x stop mix (5 µl for the above reaction), incubate at 70°C for 20 minutes and spin dialyze to remove excess reactants.

  4. 10x CIAP buffer:
    0.5 M Tris
    10mM MgCl2
    1mM ZnCl2
    Adjust to pH 9.3

    10x SAP buffer:
    50 nM Tris
    10 mM MgCl2
    Adjust to pH 9.0

    TE Buffer:
    working...
    Optional dephosphorylation:

    • Incubate the spin dialyzed plasmid with calf intestine alkaline phosphatase (CIAP) or shrimp alkaline phosphatase (SAP) at 37°C for 30 minutes:

      20 µl Spin dialyzed DNA
      2µl CIAP (or SAP)
      2.5 µl 10x CIAP buffer (or SAP buffer)

    • Incubate at 75°C for 20 minutes to inactivate the phosphatase.

  5. Add TE buffer to increase the volume to a total of 100 µl.

  6. Add 100 µl of phenol, vortex and spin in a microcentrifuge for 4 minutes. Transfer the aqueous upper layer to a new Eppendorf tube.

  7. Add 100 µl of chloroform, vortex and spin in a microcentrifuge for 4 minutes. Transfer the aqueous upper layer to a new Eppendorf tube. Repeat once.

  8. To precipitate the plasmid DNA, add 30 µl of 3 M NaOAc and 300 µl of chilled absolute ethanol. Incubate for 20 minutes at -20°C. Pellet the plasmid DNA by centrifugation for 5 minutes.

  9. Remove supernatant and wash the pellet with 1 mL 70% ethanol. Centrifuge for 4 minutes and remove the supernatant. Repeat once.

  10. After removing the supernatant the second time, invert the tube on a paper towel to blot the residual ethanol, and allow to air dry by leaving the tube on its side on the lab bench for a few minutes (do not allow to overdry).

  11. Disolve the plasmid DNA in 20 µl TE buffer. It is now ready for the ligation reaction.

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Using Restriction Endonucleases

In order to insert our DNA sequence of interest into a plasmid, we need a means to cut the plasmid at a specific site that allows correct insertion and later replication of both vector and insert. Restriction endunucleases are used for this purpose. There is a wide variety of enzymes available. Factors to be considered when using restriction enzymes include:

The manufactrer will generally provide the correct buffer and temperature for each enzyme.

Each restriction endonuclease requires a specific buffer with an optimal salt concentration, since different enzymes will need high, medium or low salt concentrations:

High ~ 100 mM NaCl Tris

Medium ~ 50 mM NaCl Tris

Low ~ 10 mM NaCl Tris

Under salt concentrations different than optimal, restriction endonucleases may recognize a different sequence. For example, EcoRI normaly requires hig salt conxcentration, and at low salt will recognize 5'--- AATT---3' instead of its usual sequence 5'--- GAATTC---3'. This phenomenom is known as star activity and denoted *EcoRI.

Restriction endonucleases work best at the optimal temperature of the organism of origin. EcoRI works best around 37°C, SalI is derived from fungy and works better at 25°C because that is the optimal temperature for fungi. Taq1 is derived from hot springs bacteria and works best at 75°C.

Reaction volume is important because bacteria are stored in glycerol, which inhibit the cutting reaction. Thus the bacteria must be diluted about x10, usually 2 µl into 20 µl.

The probability of cut depends on the number of nucleotides in the restriction enzyme's recognition sequence. For example, if the recognition sequence is 4 base pairs long and the probability of a particular nucleotide being the one recognized at a particular position is 1/4, the overall probability of recognizing the 4-nucleotide sequence is 1/256. Likewise, the probability of recognizing a 6-nucleotide site is 1/4096, and of recognizing an 8-nucleotide site is 1/65,536:

5' -- A   G   C   T -- 3'
3' -- T   C   G   A -- 5'
    1/4 x 1/4 x 1/4 x 1/4 = 1/256 		5' -- A   G   C   T   G -- 3'
3' -- T   C   G   A   C -- 5'
     1/4 x 1/4 x 1/4 x 1/4 x 1/4 = 1/4096 		5' -- A   G   C   T   G   T -- 3'
3' -- T   C   G   A   C   A -- 5'
     1/4 x 1/4 x 1/4 x 1/4 x 1/4 x 1/4 = 1/65536

In other words, these restriction enzymes will cut once every 256 nucleotides, once every 4,096 nucleotides, and once every 65,536 nucleotides respectively. Thus the longer the recognition sequence, the more specific the cutting and the lower the probability of cutting.

Another two things to consider when working with restriction endonucleases are storage and units. They must be stored at constant temperature (do not use frost-free freezers). Restriction endonucleases are not measured by weigth but in units: one unit cuts 1 µg of DNA in one hour under optimal conditions. For example, both lambda phage and the plasmid pBR322 have one EcoRI cut site. But lambda is 10 times bigger than pBR322, therefore 1µg of lambda is about 100 molecules while 1 µg of pBR322 is about 1000 molecules. So one unit of EcoRI will cut 1000 pBR322 molecules but only 100 lambda phage molecules.

Example Procedure: Cloning Using a Plasmid Vector
Part 2: Preparation of Insert

To prepare the insert, it may be necesary to cut it out from another vector or other larger piece of DNA using restriction endonucleases. The larger DNA is digested with a restriction endonuclease that ONLY cuts before and after our sequence of interest. A stop mix containing EDTA, SDS, glycerol and dye is used to stop the nuclease by chelating divalent metals needed for the reaction.

  1. Incubate 2 µg or more of DNA with the enzyme and a buffer specific for that enzyme at the recomended temperature for at least 1 hours or evernigth. An example reaction mixture follows:

    20 µl DNA
    4 µl buffer
    3 µl enzyme (30 U)

  2. To verify that the insert has been liberated, run about 1 µl of the reaction mixture, mixed with an equal volume of 5x stop mix on a 1% agarose gel along with a molecular weight marker. The 5x stop mix contains EDTA (also SDS, glycerol and dye), which stops the nuclease reaction by chelating divalent metals needed for the reaction.

  3. If the insert was compleatelly liverated, load the remaining reaction mixture, mixed with about 1/4 - 1/3 its volume of 5x stop mix, in as many wells as necessary on a 1% agarose gel with large wells and electrophorese at 100 V.

  4. When the dye reaches the bottom of the gel, identify the band for our insert and cut it out using a scalpel. The insert should be the band closest to the bottom of the gel, or if the number of bases is know, it van be identified using a weigth marker. The insert DNA can be extracted from the agarose by either resin extraction or electroelution.

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Selection Genes

The minimum requirements for a vector to be useful for cloning are a replication origin and a means of selecting recombinant vectors (have taken our insert) from wild type (have not taken our inert).

Several selection genes may be combined in one vector. For example: one selection gene may indicate if the E. coli cells were succesfully transformed while another gene indicates if the plasmids in the transformed E. coli are recombinant or wild type. Two selections genes commonly used in plasmid vectors are antibiotic resistance and the lac operon.

The lac operon contains several genes responsible for the ability to use lactose as energy when glucose is not available. The complete operon is composed of genes that code for the enzyme that breaks down lactose (beta-galactosidase), a permease that allows lactose inside the cell, and thiogalactosidase acetyl transferase (gets rid of lactose-like molecules). The lac operon also contains two promoters (P andf Pi), a represor (i), and an operator (O). When glucose supply is adequate and there is no lactose available, transcription starts at promoter for the repressor gene (Pi). The repressor protein then binds to the operator region of the lac operon and prevents transcription of the rest of the operon.

If lactose becomes available but the glucose supply is still adequate, a lactose metabolite, allolactose, binds to the represor and prevents it from blocking the path of polymerase. Still, the rest of the operon is transcribed very little because the strand has low affinity for RNA polymerase, and the enzyme detaches frequently.

When glucose becomes scarse, cAMP levels increase allowing it to bind the cAMP receptor protein, CRP (also known as catabolite activator protein, CAP). The cAMP/CRP complex can now bind to the operator of the lac operon and increases its affinity fo RNA polymerase, allowing the transcription of the whole operon.

Beta-galactosidase also converts the X-gal dye (5-Br-4-Cl-3-indolyl) to a blue color, se the lacZ gene can be used to select recombinant vectors.

                                      beta-gal
5-Br-4-Cl-3-indolyl 5-Br-4-Cl-3-indigo

For this system to work as a means to select recombinant vectors, the insert must be put in the middle of the lacZ gene. Recombinant plasmids will not turn blue upon addition of the derepresor and removal of glucose. ITPG (isopropyl-beta-gallactopyranoside) is used as a derepressor because it cannot be converted to glucose.

Most vectors have at least two selection genes because we need to determine if:

  1. the bacterial host took in the vector
  2. the vector is recombinant or wild-type

After transforming the bacterial host, they are treated with any necessary additive (for example IPTG) and plated, usually in antibiotic-treated agar, also contaioning X-gal dye. If we used a vector with genes for resistance to the antibiotic in the agar, only bacteria that took in the vector will grow. If we inserted our DNA of interest into the lacZ gene, blue colonies indicate wild-type plasmids that did not took in our insert.

Another selection method involves using two different antibiotic resistant genes in the plasmid, for example chloramphenicol (CAM) and tetracyclin (TET). If our DNA of interest is inserted into the CAM gene, we can select colonies growing in TET treated agarose but not in CAM treated agarose (see procedure below for details).

A probe may also be used to screen colonies for recombinant plasmids. [MISSING DETAILS].

"Live or death" vectors may also be used which contain a cytotoxin gene and an antibiotic. If the insert is put in the middle of the cytotoxin gene, any colonies growing in the antibiotic treated plate have recombinant plasmids.

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Transformed Bacteria

Transformed bacteria are plated to allow colonies to grow which can be selected for the prescence of the recombinant plasmid. The are plated in a 1.5% agar dish, usually treated beforehand to match the selection system being use (antibiotic resistance, beta-gal activated dye, etc). Depending on the protocol, plating may be done by diverse methods:

After the colonies have grown and are selected, they can be transfered to growing media using an inoculation stick. Aseptic techniques should be used at all times when growing bacteria to avoid contamination.

Transformed bacteria grow in liquid media in phases, as shown in the graph. The firs grow slowly, until a threshol population is reached, then a log/exponential growing phase starts. Eventually, growth reaches a stationary phase and the population almost stops increasing. In order to induce the growth of plasmid, bacterial growth must be stoped somewhere in the log/exponential, when there is a lot of bacteria and still nutriens left to sustain plasmid replication. This is done by adding an inhibitor when the optical density (OD) of the mixture is about 0.6-1.0 as measured in the mass spectrometer.

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