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BIOLOGY 1020 ASSIGNMENT #3 – Human-Engineered Plasmids
Why Plasmids?
Plasmids are circular rings of double-stranded DNA that are used in prokaryotic cells to store additional genes. These
plasmids can be shared from one bacteria to another in the process of conjugation. This is one of the few ways that
living things can spread new genes, and thus new traits, within the same generation. For most life on Earth, new gene
combinations can only occur in the next generation, after reproduction has occurred. In nature, bacteria can share genes
that code for any number of traits via plasmids, however, plasmids that code for antibiotic-resistance or allow new food
sources to be processed are among that most powerful plasmids.
The process of conjugation. A plasmid in the donor cell is duplicated and copied to the recipient cell via a conjugation
pilus. Once conjugation is completed, both cells now contain the plasmid and can express all the genes it contains.
Plasmids are useful for scientists because they can be used to introduce new genes into bacteria without editing their
main genome (their main strand of DNA). Attempting to edit the bacteria’s main DNA is very difficult as it is very much
larger and more complex than a plasmid. Editing a plasmid is similar to building a storage shed next to a house – it has
its challenges, but the work is much simpler than trying to add a new room to the existing house.
Plasmids can also be easily added to a bacteria, while attempting to edit the existing DNA would be much more
complicated (having the remove the DNA, edit it, and then place it back in). Plasmids can also be mass-produced and
introduced into bacteria as a group. Editing the DNA of each bacteria individually would take significantly more time,
effort and money. Given that many experiments require trial and error when adding new elements to a plasmid, editing
the individual cells’ DNA would prove extremely time-consuming.
1)
What is a plasmid and what do bacteria use them for in nature?
2)
Why do scientists prefer to use plasmids instead of editing the bacteria’s main DNA?
DNA Ligase and Restriction Enzymes
To make a plasmid, scientists need to be able to cut apart and reassemble pieces of DNA. The process of taking two
pieces of DNA and joining them is called recombination (and DNA that has been assembled from two or more pieces is
called recombinant DNA). Recombination occurs in nature, and is an important part of many life processes.
Assembling DNA is a relatively easy process. Scientists use a type of enzyme known as a DNA ligase to “glue” two pieces
of DNA together. DNA ligase is a type of enzyme that exists in living things and does the job of attaching pieces of DNA
together – scientists have simply isolated it and put it to work.
DNA Ligase assembling two pieces of double-stranded DNA together.
Cutting DNA molecules is a bit more challenging. DNA molecules are long and complex chains but are also extremely
small compared to the things people work with in their everyday lives. DNA is much too small to cut with a knife, for
example. There are chemicals and enzymes that can break down DNA, such as the ones produced by the stomach during
digestion, but these cut DNA at all of the bonds, making them useless for cutting the DNA at specific locations.
Luckily, there exist in nature a series of enzymes that cut DNA with a very high degree of specificity. These enzymes are
known as restriction enzymes and will cleave DNA molecules at a very specific sequence. Each restriction enzyme
targets a different sequence, known as its restriction site. Over the years, scientists have built up a library of restriction
enzymes that they have isolated from various creatures to allow them to cut many different pieces of DNA in different
places.
The restriction enzyme EcoRI looks for the specific sequence GAATTC and cuts it into G and AATTC fragments.
Scientists can use the various restriction enzymes available to cut DNA exactly where they want it cut. This avoids cutting
up regions of DNA with genes that a scientist needs intact. A scientist carefully chooses their restriction enzymes so that
they cut around the good bits of DNA.
Restriction Enzyme sites are chosen carefully to cut before and after the useful gene. Sometimes useful genes can contain
the target site for a restriction enzyme and so a scientist may have to go through several restriction enzymes before
finding one that cuts before and after a useful gene, but not within the gene.
3)
What is recombinant DNA? Is all recombinant DNA human-made?
4)
How do scientists attach two pieces of DNA together?
5)
Chemicals such as hydrochloric acid and enzymes such as pepsin are produced by your stomach and break down
the DNA in the food you eat. Even though these methods can break down DNA, why are they not suitable for
creating plasmids?
6)
What is a restriction enzyme?
7)
Why do scientists need more than one restriction enzyme to make plasmids?
Antibiotic Resistance in Plasmids
When a plasmid is built and introduced to a group of bacteria, not all of the bacteria will take up the plasmid. However,
the scientists will want to isolate only the plasmid-carrying bacteria for the next steps of their work. Looking at the
bacteria individually and sorting them is complicated, difficult, and very time-consuming. Therefore, scientists have
developed ways to filter out all of the bacteria missing the plasmid.
One of these methods involves using an antibiotic resistance gene in the plasmid. When the plasmid is made, the
scientist adds a gene that makes the bacteria resistant to a specific antibiotic (a chemical that kills bacteria). This means
that bacteria with the plasmid can survive if this antibiotic is present, while the bacteria without the plasmid will die.
After the plasmid has been forced into the bacteria, the bacteria are put onto a plate containing food and the antibiotic.
Any bacteria that took up the plasmid will survive and multiply on the plates, while bacteria lacking the plasmid will die.
After some time has passed, the only living bacteria on the plate will contain the working version of the plasmid and can
be taken from there to be used in the next step of the work the scientist is doing.
An added bonus to using antibody resistance genes in the plasmid is that the antibiotic will kill off any other bacteria
that get onto the plate from the air or other sources. This helps eliminate other bacteria from getting into the mix and
disrupting the experiment. As well, sometimes the process of assembling a plasmid can go wrong. By using an antibiotic
gene in the plasmid, the scientist will ensure that not only do all the bacteria left living at the end have the plasmid, but
that all of the bacteria have a properly-assembled and working plasmid.
If antibiotic resistance is not used, other genes can fill the same role, such as genes that allow a bacterium to live on a
new food source. In that case, bacteria are plated on a medium with the only food being the one that requires the
plasmid’s genes to digest. Again, this results in the only bacteria alive at the end having a working version of the plasmid.
8)
What is the purpose of adding an antibiotic resistance gene to plasmids?