Joining, transferring and selecting the recombinants ·… | StemPy Lab
Course 27.2·Lesson 3 of 3
Joining, transferring and selecting the recombinants
The cut vector and the gene are joined by ligase, and the recombinant DNA is put into a host. A bacterium must be made competent, by calcium and a heat shock. Animal and plant cells take DNA by microinjection, a gene gun or a disarmed vector. Cells that took the DNA are found by a selectable marker. Recombinants are found by insertional inactivation.
about 30 min1 simulation1 practice question
Lesson 3 of 3Show all lessonsHide
Try the code and the simulations beside the lesson.
Join and deliver
You now have a vector cut open and a gene with matching ends, so the next job is to join them. DNA ligase is added to the mixture. It joins the fitting sticky ends and makes the recombinant DNA. Two more jobs remain. First, the recombinant DNA must get into a host cell. Second, we must find out which cells took it up.
Getting the DNA into the host
DNA is a large molecule and it likes water, so it cannot slip through the membrane of a cell. There are several ways to help it. The best method depends on the host.
Four small panels. Panel 1, heat shock: a sketch of temperature against time in which competent cells with the recombinant DNA are on ice, then briefly at 42 degrees Celsius, and then on ice again. Panel 2, microinjection: a fine needle injects the DNA straight into the nucleus of an animal cell. Panel 3, gene gun: tiny gold or tungsten particles coated with DNA are shot at high speed into a plant cell. Panel 4, disarmed pathogen vector: a disarmed Agrobacterium or retrovirus carries the DNA into a plant or animal cell.
A bacterium. The cell must be made competent, which means ready to take up DNA. The steps are these.
The cells are treated with a particular concentration of a divalent cation, such as calcium. This increases the efficiency with which DNA enters the cell through pores in its wall.
The cells are kept with the recombinant DNA on ice.
The cells are given a brief heat shock at 42 degrees Celsius.
The cells are put back on ice. The bacteria have now taken up the recombinant DNA.
An animal cell. In microinjection, the recombinant DNA is injected with a very fine needle directly into the nucleus of the cell.
A plant cell. In the biolistics method, or gene gun, the cells are bombarded with gold or tungsten particles coated with the DNA, which are shot at a high speed.
A disarmed pathogen vector. A pathogen is changed, so that it can no longer cause disease, and is used as a carrier. It infects the cell and puts the recombinant DNA inside. The Ti plasmid of the bacterium Agrobacterium tumefaciens is changed in this way for plants, and retroviruses for animal cells.
Finding the cells that took the DNA
After the treatment, only a small share of the cells has taken the recombinant DNA. The others have nothing. We must remove them. The selectable marker does this job. In pBR322, there are two, ampR and tetR. Suppose the cells are plated on a medium that contains ampicillin. The cells with no plasmid die. The cells that took up the plasmid, with or without an insert, grow, because they have ampR. So ampicillin picks out the transformants.
Finding the recombinants: insertional inactivation
A transformant can have a plasmid with the foreign gene (a recombinant), or a plasmid that closed again without any insert (a non-recombinant). They look the same on the ampicillin plate. We need another test.
Insertional inactivation is the idea that a gene is switched off when a foreign DNA is inserted into it. Suppose the foreign gene was ligated into the BamHI site, which lies inside tetR. The gene is now broken, and the cell is no longer resistant to tetracycline. It is still resistant to ampicillin, because ampR is whole.
Two round plates seen from above, and a result box. The left plate holds nutrient agar with ampicillin, and ten colonies have grown on it. The right plate holds nutrient agar with tetracycline and is a replica of the left one. Only four colonies have grown on it, at the same places as four of the first ten. The other six places are shown as dashed empty circles. The result box says that the six colonies that grew on ampicillin only are the recombinants, because the foreign DNA broke the tetR gene, and the four that grew on both carry a plasmid without an insert.
No plasmid: dies on ampicillin, dies on tetracycline.
Plasmid without an insert: grows on ampicillin, grows on tetracycline.
Recombinant plasmid, insert in tetR: grows on ampicillin, dies on tetracycline.
The usual way is to spread the cells on ampicillin, and then to press a copy of the plate on tetracycline (a replica plate). The colonies that grew on the first plate and did not grow on the second are the recombinants.
Blue and white colonies
There is a different marker, which is easier to read. Some vectors carry the gene lacZ, which codes for the enzyme beta-galactosidase. The cloning site lies inside this gene. When the cells are grown on a medium with a chromogenic substrate, a substance that gives a colour when it is broken down, a working enzyme makes the colony blue.
A plasmid without an insert has a working enzyme. The colony is blue.
A plasmid with the foreign DNA in lacZ has no working enzyme. The colony stays white, with no colour. These are the recombinants.
On the left, a round plate with twenty colonies, five of them blue and fifteen of them white. A key says that blue colonies have a working lacZ gene, so the enzyme turns the chromogenic substrate blue, and they carry no insert. White colonies have a lacZ gene broken by the foreign DNA, and they are the recombinants. On the right, a small circle that stands for the plasmid shows the gene lacZ with the cloning site inside it, and an arrow from the foreign DNA to this site.
The substance X-gal and the inducer IPTG are used in this test in laboratories. NCERT does not name them. They are taught at A-Level and IB.
The principle, in one line
The marker is placed so that the insertion of the gene breaks it. A broken marker tells you that the gene went in.
You should see that when most colonies on the ampicillin plate also grow on the tetracycline plate, few recombinants are present. The recombinants are the difference between the two counts.
Try it in Python
The program writes the table of the three kinds of cell and the two kinds of plate.
Practise with new numbers
Common mistakes
Saying that ampicillin finds the recombinants. Ampicillin finds the transformants. The recombinants are found by the broken marker.
Mixing up the colours. Blue colonies have no insert. White colonies are the recombinants.
Forgetting that the cells must be competent. A bacterium does not take DNA without a treatment.
Mixing up the methods. Microinjection is for the nucleus of an animal cell. The gene gun is for plant cells.
Saying that the heat shock is the main treatment. The calcium makes the cell ready. The heat shock helps the DNA enter.
Remember these
Bacteria: calcium (divalent cation), recombinant DNA on ice, a brief heat shock at 42 degrees Celsius, back on ice.
Animal cell: microinjection into the nucleus. Plant cell: the gene gun with gold or tungsten particles. Disarmed pathogens: Agrobacterium and retroviruses.
Selectable marker: finds the transformants. Insertional inactivation: finds the recombinants.
BamHI inside tetR: recombinants grow on ampicillin and not on tetracycline. lacZ: recombinants are white.
Check yourself
Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.
Describe how a bacterium is made competent, and how it is given the DNA.
Which method puts DNA into the nucleus of an animal cell, and which one is used for plant cells?
What is insertional inactivation?
A foreign gene is ligated into the BamHI site of pBR322. Which plates do the recombinants grow on?
On a plate with a chromogenic substrate, 6 colonies are blue and 30 are white. How many colonies are recombinants, and why?
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 17.2.
Mandel M. and Higa A. (1970). Calcium-dependent bacteriophage DNA infection. Journal of Molecular Biology 53, 159 to 162.
Enroll free in Making a Recombinant DNA, Step by Step
Every lesson is open to read. Enrolling also lets you:
Mark lessons done and carry on where you stopped.
Take the tests of this lesson and its course.
Count the course towards the certificate for its chapter.