Recombinant DNA

Also Known As…

No, we won’t be making the elusive dog-boy or elephant-crocodile in the lab…So please

stop asking.

WHAT IS “RECOMBINANT” DNA?

• We already know what DNA is – the genetic blueprint and all – so let’s focus on the recombinant part of it.

• “Recombinant” refers to the fact that a sample of DNA has DNA originating from two different sources or organisms.

• Basically, we are taking a gene for a trait from one organism and inserting it into the genome of a second organism to see if it can be adopted and used.

• If successful, the second organism can produce the protein that the inserted, foreign gene is responsible for making.

Step 1: CUT IT OUT

• The first thing we must do is extract the desired gene (that we wish to insert into something else) from the source organism.

• We can isolate the DNA from the source and use a restriction enzyme such as EcoR I to cut out the gene. We must make sure there are cut sites close to the ends of the gene but not in the gene – we don’t want to cut the gene in half or it is useless.

• As this is being done, a plasmid from a bacterial cell is being selected, isolated and copied. The plasmid will also have a recognition site for the restriction enzyme used for the foreign gene.

Step 2: INSERT INTO VECTOR

• The bacterial plasmid is our vector for the foreign gene. This means the plasmid is the mode of delivery for the foreign gene into the host cell.

• Viruses are another vector being considered for this role in biotechnology.

• We want the foreign gene to use the sticky ends produced in step 1 to insert itself into the plasmid.

• Ligase is added in order to seal the breaks in the DNA backbone.

• This will leave use with several outcomes:• Normal plasmid…no…• Two plasmids attached…nope…• Genes that have attached together…sorry…• A recombinant plasmid – we want this!

Inserting Foreign DNA into a Plasmid

Step 3: Transformation

• Now we run a process called a transformation in order to get the recombinant plasmids into the bacterial cells.

• The bacterial host cells are manipulated in order to make them more permeable to adoption of the plasmids. This can be done using electroporators, gene guns or chemicals such as calcium chloride.

• Once the bacterial cell takes up the recombinant plasmid, it is referred to as being transformed.

• All of the surviving cells are cultured in the lab and a COPY is made of this plate!!!

• The only thing is you don’t know what plasmid has been taken up…the recombinant one or the normal, unaltered plasmid.

Step 4: TEST FOR COMPETENCY• Now we have to be able to discern between the

following:• Cell with no plasmid…no…• Cell with non-recombinant plasmid…nope…• Cell with recombinant plasmid in it…BINGO!

• The cell with the recombinant plasmid is called a competent cell. We want this but we have to be able to pick it our from the other two cell types.

Step 4: TEST FOR COMPETENCY

• To perform this testing process we must first look at the plasmid in detail…

• A plasmid is “bonus” DNA – non-essential to bacterial cell function but beneficial in that it carries resistance genes to potential threats to the cell.

• We will use these properties in order to identify our competent cells. How?• The foreign gene is inserted into one of the

resistance genes in the plasmid and interrupts the function of that gene – removing the resistance!

Step 4: TEST FOR COMPETENCY

• Look at the plasmid on the right as an example…

• If we cut this plasmid with PST I and insert the foreign gene within the Ampicillin resistance gene, the cell is no longer resistant to ampicillin.

• The tetracycline gene is not affected in any way so the cell with the plasmid in either form is still resistant to tetracycline.

• We can use this information to help us identify competent cells!

Step 4: TEST FOR COMPETENCY

• Keeping our plasmid in mind…• Along with our three cell types…

• First lets treat the cells with Tetracycline!• Second lets treat the remaining cells with Ampicillin!• Nooooooooooo! We just killed the cells we wanted!• But wait! We made a copy of the bacterial colonies

before the testing!

Step 4: TEST FOR COMPETENCY

• This is what the bacterial plate might look like during testing…can you tell which colonies were formed by competent cells?

• Test 1: Tetracycline!• Test 2: Ampicillin!• Obviously, colonies

2 & 4 have the

recombinant plasmid

inside of them.• No go back to the copy

of this plate and grow them!

Step 5: GROW BACTERIA AND CASH!!!

• Once you have grown many copies of the recombinant bacterial cultures you can harvest the desired product of the foreign gene and make lots of money because bacteria work for super cheap!

FIN