HSC Biology: Recombinant DNA Technology

Written by Simon Tang.

Key Points Summary

• The gene of interest and the plasmid vector are combined using restriction enzymes
• The plasmid vector will be inserted (transformed) into the bacteria via heat shock or electroporation
• Combining the gene of interest and vector is not 100% accurate, and neither is bacterial transformation. Thus, we need to somehow select for bacteria that have the plasmid with the gene of interest.
• Vectors have a gene that makes bacteria resistant to an antibiotic. Thus, if you grow your bacteria on a petri dish with that antibiotic, only bacteria with the plasmid can grow.
• If the gene of interest was successfully combined into the plasmid, the reporter gene would be interrupted and would not work. Usually, the reporter gene is lacZ, and in the presence of X-gal, it would make a blue dye. Thus, any bacterial colonies that aren’t blue (white) will have the gene of interest.

The Introduction

What is recombinant DNA technology? As one of the last syllabus points in Module 6 (Genetic Change), HSC Biology students are faced with trying to understand this complex process, and how it applies to society today. Here, I hope to break it down into manageable steps, using bacteria as our example.

The Gist

According to the NCBI, recombinant DNA technology “enables individual fragments of DNA from any genome to be inserted into vector DNA molecules, such as plasmids, and individually amplified in bacteria.” Basically, you can take a gene from any organism, animal, plant, fungi, and put it into a bacterium. By putting it into a bacterium, as the bacteria divides and continues to grow, you will have more of that gene, since it will duplicate each time the bacteria divides.

The reason why this is important is because it gives us a way to make many copies of any gene we want, as well as its protein product. Remember that a gene codes for a polypeptide sequence (which can make a protein). There are many diseases out there in the world that are caused by a faulty gene, or a lack of a specific protein product. A common example is diabetes, which is when people cannot produce the protein product insulin to regulate sugar levels. By using recombinant bacteria to produce human insulin for us, and then giving it to diabetic people, we can help them lead long and happy lives.

The Nitty-Gritty

As mentioned above, the way recombinant DNA technology works is by inserting a gene from any organism into bacteria, and then letting the bacteria to grow to produce many copies of the gene and its protein product. However, there are many parts to this process. This section breaks them down into detail.

1. The Vector

To put the transgene (an artificially introduced gene) into the bacteria, we need a vector to carry it into it. This comes in the form of a plasmid.

A plasmid is basically a small circular section of DNA that bacteria already naturally have in their cells, so it will be easier for them to take up our man-made ones. Scientists use a special type of vector that contains many essential elements, shown above. Why these elements exist will be explained in Step 4.

2. Putting Gene of Interest into Vector

The way that scientists put the gene of interest into these vectors (currently referred to as empty vectors) is by using enzymes called restriction enzymes.

These enzymes cut at a very specific location of the empty vector and the gene is inserted at that location. The empty vector is designed so that the place where the gene of interest is inserted is right in the middle of the reporter gene, so the reporter gene will no longer work. 

When the gene is inserted, the empty vector becomes a recombinant plasmid. This process is not 100% accurate though, so by the end of it, you will have a mixture of empty vectors and recombinant plasmids.

3. Transforming the Vectors into the Bacteria

Now that we have our plasmids, it is necessary to insert it into the bacterial cells – this process is called transformation. The most common method is heat shock, where bacterial cells are mixed with the plasmids and heated to 42°C for about a minute. The heat causes the cell membranes to be disrupted just enough for the plasmids to enter.

Just like before, this process is not perfect – not every bacterium has a plasmid inserted into it. This means that we have three types of cells in our mix:

Cells with no plasmid

Cells with a plasmid without the gene of interest

Cells with a plasmid with a gene of interest

We only want the last type.

4. Screening the Transformants

As the final step, we now need to remove all the bacteria that either 1) do not have a plasmid in them or 2) have the empty vector instead of the recombinant plasmid. We just want a pure strain of the bacteria with the recombinant plasmids to make our protein products.

To achieve this, we need to take advantage of some of the elements of our empty vectors.

This is done in two steps:

a. Antibiotics: Recall that in the empty vector diagram above, the green section of the plasmid is a gene for antibiotics resistance. That means that every cell that has either the empty or the recombinant plasmid is going to be resistant against antibiotics (which normally kill bacteria).

Thus, if we grow these bacteria on a petri dish with some antibiotics on it, voilà! All the cells with NO plasmids will all die. One down, one to go.

b. Reporter genes: Recall that when the gene of interest was inserted into the plasmid, it interrupted a reporter gene called lacZ.

We can use this to help distinguish between bacteria with the empty and recombinant plasmid.

When growing bacteria on the petri dish, on top of antibiotics, we can add a second ingredient called X-gal. What happens is that all the bacteria with a functioning lacZ gene (the bacteria with the empty vectors) will produce a protein product that will react with the X-gal and create a blue dye. Thus, bacterial colonies transformed with empty vectors appear blue.

In the recombinant plasmid, the lacZ gene is disrupted by the gene of interest. It is unable to be expressed into its protein product, and hence X-gal cannot be turned into its blue product. Hence, recombinant bacterial colonies appear white. This is called blue-white screening.

Thus, by simply scraping off a white bacterial colony and isolating it somewhere else, we have just E. Coli with the gene of interest in them. Complete!

The Wrap-up

In short, recombinant DNA technology is a way for humans to make many copies of genes and its protein products using bacteria. It can be used to produce invaluable protein products to treat diseases (like producing insulin to treat diabetes), and saves millions of lives every year.