Molecular Cloning - Part I (Introduction and Vector Selection)

Introduction -

A clone means a genetically identical copy of the original. Cloning is a naturally occurring process in bacteria, fungi, and some plants that undergo asexual division (i.e., nature’s way of ctrl C and ctrl V). In humans, identical twins are an example of sexual division resulting in clones, as the original fertilized egg splits, resulting in two or more genetically identical embryos. This process was studied and refined into techniques for artificially generating clones. Artificial cloning is of 3 types based on the level at which it is performed: molecular, cell, and organism cloning. Artificial cloning provides scientists with a powerful tool to study mutational impacts in fields such as medicine, especially in cancer research (molecular cloning). It also allows for the protection of endangered species (organism cloning), to create plant and animal strains that provide higher yields and are resistant to harsh environments as well as diseases (cell/organism cloning). Cloning also helps create patient stem cell-derived tissues for transplanting damaged tissues (cell cloning).

In this article, we are interested to understand molecular cloning, a part of recombinant DNA technology used in several research labs.

Molecular cloning

As the name suggests, it is the technique of creating clones of molecules, mainly DNA or RNA. This is primarily used in research to identify gene regulatory regions or to understand some genes’ mutational burden. In this technique, specific genes from the organism’s genome are cloned and introduced into systems such as plasmids (extra-nuclear DNA found in bacteria), which express the cloned gene under particular conditions.

This technique involves steps such as:

1. Amplification: The genes or regions of interest are concentrated and collected using a combination of PCR (polymerase chain reaction) and Agarose gel electrophoresis.

2. Fragmentation: The amplified DNA and the desired plasmid are cleaved using the same restriction enzymes to facilitate the ligation process by creating sticky ends.

3. Ligation: This is the process of gluing/stitching the amplified fragments to the plasmid that has also undergone restriction digestion. The gluing/stitching occurs at the sticky ends.

4. Transformation/Transfection: The ligated plasmid is then introduced into bacteria (transformation) or mammalian cells (transfection).

5. Screening/Selection: This method selects the cells that have successfully taken in the plasmid.

6. Sequencing: This method is used to confirm the sequence of the amplified and restricted DNA to ensure the identity of the gene or segment that has been successfully cloned.

Vector selection -

Vector is the vehicle DNA that carries the gene of interest (foreign DNA) into the host. There are two types of vectors:

• Cloning vector - Used to incorporate Gene of interest (GOI) into a host.

• Expression vector - Not only for cloning the GOI but also for assaying its relevant function as protein analysis.

Important criteria for Vector Selection –

1. Origin of replication –

The cloning vector should have its origin of replication for autonomous replication of the GOI using the host’s machinery.

2. Multiple Cloning Site(MCS)/restriction site –

A plasmid/vector can have multiple restriction sites in its MCS. Selecting a restriction site involves two steps –

(a) Prevent formation of blunt ends – When a restriction enzyme introduces a straight cut without any overhangs (or unpaired bases), it results in blunt ends. Due to difficulty in getting the correct orientation of GOI and ligation, blunt ends are generally not preferred. Also, it’s recommended to use two different restriction enzymes cutting at 5’ and 3’ end to prevent the change of GOI orientation.

(b) Check the sequence of GOI – The GOI can also carry a region where a restriction site acts or cuts the sequence. Before selecting the restriction enzymes, run the sequence through a server referred to as zero cutter, which will provide a list of restriction enzymes that can act on the GOI and, therefore, should not be used.

*It’s always a good practice to check the availability of the enzyme in your respective labs to prevent the end-time hassle.

3. Selectable markers -

Once the recombinant DNA gets inserted into the host organism, it is said to be transformed. Not all the cells within the host will contain recombinant DNA. Therefore, selectable markers act as ‘genetic tags’ to select the positively modified cells. A few examples are antibiotic-maker genes and metabolic/auxotrophic genes.

4. Expression system –

The most important criterion while selecting an expression vector is the organism/host cells in which the GOI is to be expressed. As per the experimental requirement, the expression system can vary. A bacterial expression system is preferable for smaller-sized genes encoding smaller proteins. An insect-based or mammalian expression system is useful for larger genes, protein-complex, or proteins requiring post-translational modifications (PTMs) for proper folding or activation. Further, studying proteins secreted by the cells or for experiments such as protein-protein interaction yeast expression system can be used.

Here are some examples of commonly used vectors for different expression systems –

· Bacterial – pGEX-KT, pET28a, pNIC-CH

· Yeast – pPIC

· Mammalian – pUC19

· Baculovirus – pFASTBac

· Plant - pCambia

5. Tags –

Tags are peptides used to purify/detect the protein of interest (POI).

• Affinity – An affinity tag is used to purify POI from a pool of other cellular proteins (referred to as junk). For example, His tag (sequence of 6-8 histidine residues) has an affinity toward Nickel (Ni). Therefore, Ni-NTA beads are used to purify proteins expressed with His tag. Other examples are GST (Glutathione-S Transferase) tag with an affinity to GSH (Glutathione), Biotin to Streptavidin, and MBP (Maltose-binding protein) to Amylase.

• Solubility – Some proteins can be challenging to purify majorly because they do not fold properly and therefore are not soluble in the given expression system. Some tags, such as Thioredoxin (Trx), GST, and MBP, can be used to solubilize these proteins.

• Detection – Obtaining a readily available specific antibody for a particular protein may be difficult at times. Therefore, tag antibodies can be used to detect POI or pull down a pool of proteins bound to the POI. Examples of epitope tags can be – the FLAG tag (DYKDDDDK), Myc tag (EQKLISEEDL), and HA tag (YPYDVPDYA).

• Fluorescence – Fluorescent tags are particularly used to study the localization of proteins in live or fixed cells. It can also be used sometimes to quantify the protein or transcript levels. The most common example of fluorescent tags is GFP (Green fluorescent proteins) and RFP (Red fluorescent proteins).

Content writers - Mr. Shubhashish Chakraborty and Mr. Naythan Dcunha

Edited by - Ms. Neha Mishra