Expressing the foreign gene and growing the culture

From the plate to the product

A white colony on a plate holds a recombinant plasmid. That alone does not feed anyone or cure anyone. The goal is the product: the protein that the foreign gene codes for. To get it, the host must express the gene, which means to read it and make the protein.

If any gene that codes for a protein is expressed in a heterologous host, a host of another species, the protein is called a recombinant protein. Human insulin made by a bacterium is an example. The next chapter tells the story.

Small scale and large scale

NCERT describes two stages.

• Small scale. The host cells grow in a flask in the laboratory. This is good to test whether the gene works. It makes too little product to sell.
• Large scale. To get large amounts of the protein, the cells must grow in a very large volume. This is done in a bioreactor, the subject of the next lesson.

Whatever the scale, the cells must grow, and the growth of a culture follows a pattern.

The growth curve of a batch culture

In a batch culture, the cells are put in a fixed amount of medium, and nothing is added or taken out until the end. Four phases follow.

1. Lag phase. The cells have just been put into a new medium. They adjust, and make the enzymes that they need. The number hardly changes.
2. Log phase, also called the exponential phase. The cells divide at a steady pace. On a log scale, the curve is a straight line. This is the time of fastest growth.
3. Stationary phase. Food runs short, wastes build up, and the number of cells levels off. New cells are made as fast as old cells die.
4. Decline phase. More cells die than are made, and the number falls.

The log phase is the one with a steady doubling time. For a product that is made while the cells grow, the culture is taken in or near this phase. For a product that is made when growth slows, the culture is taken later. The right time is different for each product.

A model that levels off

The simulation below uses the logistic model. It leaves out the lag phase. The number of cells $N$ after $t$ hours, with a limit $K$, is

Here $N_0$ is the number at the start, and $r$ is the rate of growth. At first the curve rises like a doubling. Near $K$, it flattens.

Expression needs more than a gene

A gene is read only if it has the signals that the host recognises. A host that makes the protein from a gene of another species needs a promoter that works in it. This part is beyond NCERT, and A-Level and IB teach it. Genes of animals have introns, which a bacterium cannot remove. So, to make a human protein in a bacterium, the workers use a DNA copy made from the processed mRNA, or a gene that is made in the laboratory.

You should see that a bigger start makes the culture reach half of its limit sooner, and a faster rate does the same. After half of the limit, the growth slows.

Try it in Python

The program uses the same model. It prints the cells at some times, and the time to reach half of the limit.

Practise with new numbers

Common mistakes

• Saying that the number of cells keeps rising. In a batch culture it levels off, and later falls.
• Mixing up the lag and the stationary phases. In both the number is steady, but the lag comes first and the cells are adjusting. In the stationary phase, food has run short.
• Saying that a recombinant protein is made by the host from its own gene. It is made from a gene of another species.
• Thinking that a flask is enough for the market. It is only the first step. A bioreactor is used for large amounts.

Remember these

• Expression is the making of the protein from the cloned gene. A protein from a gene expressed in a heterologous host is a recombinant protein.
• Small-scale culture in a flask, large-scale culture in a bioreactor.
• A batch culture: lag, log (exponential), stationary and decline phases.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. What is a recombinant protein?

2. Why is a flask culture not used to make a medicine for sale?

3. Name the four phases of the growth of a batch culture, in order.

4. In which phase does the number of cells double at a steady pace?

5. Why does a culture level off even though the cells are healthy?

References

• NCERT (2023). Biology, Textbook for Class XII, chapter 9, Biotechnology: Principles and Processes.
• Clark M. A., Douglas M. and Choi J. (2018). Biology 2e, OpenStax, chapter 17.3 (Biotechnology in medicine and agriculture) and chapter 4.
• Monod J. (1949). The growth of bacterial cultures. Annual Review of Microbiology 3, 371 to 394.