Plasmids & Plasmid Mapping
Elianna Van Raalte - 2017
Page Contents:
What are Plasmids?
The Discovery of Plasmids
Bacterial Conjugation
Scientific Uses of Plasmids
Plasmid Mapping
Definitions (words denoted throughout with asterisks will be in the Definitions section)
Bibliography
What are plasmids?
Plasmids are circular pieces of double-stranded DNA in a cell that are not in a chromosome which include information that is not necessary for a cell's basic function. Plasmids often include accessory genes that encode advantageous functions for the host cell, and can work to transfer genetic material between cells.
Usually, plasmids are found in bacterial cells, but they have also been discovered in eukaryotic* cells such as yeast and fungi.
Plasmids can replicate independently of their host cell. A cell can host from one to a thousand plasmids, depending on the number of times the plasmid replicates.
Types of plasmids:
There are five recognized types of plasmids, each of which encode the information to perform vastly different beneficial functions for their host cell.
1. Fertility plasmids - these are called 'fertile' because they can perform conjugation, passing on their information to other bacteria. (see below for more on conjugation)
2. Resistance plasmids - contain genes that are resistant against antibiotics
3. Col plasmids - encode the manufacture of proteins that kill other bacteria
4. Degradative plasmids - allow for digestion of unusual substances
5. Virulence plasmids - these turn their host cell into a pathogen*
Joshua Lederberg, 1962
Discovery of plasmids
In the 1940s-1950s, scientists knew that there were genetic factors that could be transferred between cells, but didn’t understand them and weren’t sure what to call them.
In 1946, Joshua Lederberg decided to investigate how bacteria reproduced. He found that they didn't just clone themselves - there was a sexual-like phase during which they shared genetic information.
in 1952, Lederberg introduced the term "plasmid." The term was coined to classify any genetic particles outside the chromosomes.
Lederberg won the Nobel Prize in 1958 for his work in the field of genetics.
Bacterial Conjugation
What is conjugation?
Bacterial conjugation is the sharing of genetic information between bacteria by physical contact.
Because plasmids contain genetic information and are able to move around, plasmids are essential to this process, however not all plasmids are useful for conjugation. Specific plasmids called conjugative plasmids* must be part of a donor cell in order for it to complete conjugation.
A photo of bacteria conjugating.
The picture shows Step 2 in the diagram below - the pilus drawing the bacteria together.
Why conjugate?
Plasmids can contain information that is very helpful for a bacteria cell. For example, antibiotic resistance is passed between bacteria on plasmids by conjugation. Plasmids can also carry genes that aid the bacteria in killing other bacteria or in digesting irregular substances.
This means that in stressful situations, a bacterium with a plasmid is more likely to survive than one without. The more a cell conjugates, the more cells will have those beneficial genes.
The Process of Conjugation:
- The donor cell produces a pilus.*
- The pilus connects the two bacteria and draws them together.
- The plasmid is nicked* at the origin of transfer* and one of the two strands enters the receiving cell.
- Both bacteria reform a plasmid from the single strand of information:
- The single strands form circles.
- Each cell copies a new complementary strand to become double stranded.
Both cells grow pili* and are now able to donate their information to other bacteria.
Scientific Uses of Plasmids
Plasmids can be cut open and inserted with new DNA. They will then reattach and pass that information on to other cells by conjugation. The bacteria replicates with the new instructions (ex. to build insulin). The changing of genetic information allows many different applications in genetic engineering.
Lab-modified plasmids are called vectors*.
Bacteria can be forced to keep and replicate vectors, since the vectors often carry genes for antibacterial resistance. Scientists can grow the bacteria in the presence of an antibiotic, and only those bacteria with that modified plasmid will survive.
Scientists first started using this method of DNA transfer in the 1970s. Because the method is so useful, scientists still utilize DNA transfer through plasmids in many lab experiments.
A helpful animation of a plasmid being modified.
Creating Vectors
As mentioned in the above video, restriction enzymes* cut the strands of DNA at specific sequences of information called recognition sites.* A restriction enzyme cuts sticky ends of DNA to attach together any two DNA molecules that are cut with the same restriction enzyme. An enzyme called DNA ligase* attaches the matching sticky end together.
Plasmid Mapping
Every piece of DNA can be cut into fragments by restriction enzymes.
The fragments can then be separated and organized into their specific pattern through a process called gel electrophoresis*. Different fragments of DNA with different molecular weights move through gel at a different speed, marking the gel in distinct bands.
This allows scientists to analyze them and determine the characteristics of the encoded information.
The relative distance between restriction enzymes can be determined by the information given in the gel electrophoresis. These distances are then mapped onto a circle called a restriction map.
This restriction map shows many sites of cutting by restriction enzymes.
The information given is helpful for scientists when they are planning to insert other information into the plasmid.
It can also be useful to see the restriction map of an already modified plasmid, in order to figure out the orientation of the inserted DNA.
Enjoy this rap that sums up much of the information above about plasmids.
You will notice some plasmid maps in the graphics.
*Definitions:
Conjugative plasmid - a plasmid that includes specific coding, including coding for an origin of transfer and for transfer genes. Conjugative plasmids are necessarily part of a donor cell in bacterial conjugation.
DNA ligase - an enzyme that connects matching sticky ends of cut DNA strands together.
Eukaryote - an organism that has a nucleus.
F-factor - the strand of DNA that encodes the production of a pilus, therefore allowing that cell to initiate bacterial conjugation.
Gel electrophoresis - a procedure that separates charged molecules based on their molecular weight. This process is used to organize and analyze fragments of DNA.
Nic - a cut in a DNA strand, where there is no sugar-phosphate bond. Plasmids are usually nicked to allow for the removal of one of the DNA strands during conjugation. (verb = to nick)
Origin of transfer - a section in the DNA strands where one strand is cut to allow it to transfer into a recipient cell.
Pathogen - a bacteria that causes disease.
Pilus - a tube-like structure produced by a bacterium that connects two bacteria for the purpose of conjugation. (plural = pili)
Prokaryote - an organism that does not have a nucleus, such as a bacterium.
Recognition sites - a specific base-pair sequence in a DNA molecule at which a restriction enzyme will cut. Recognition sites are usually 4-8 base pairs long.
Restriction Enzymes - enzymes that catalyze the cutting of a two-stranded DNA molecule at a specific base-pair sequence.
Transfer genes - a gene activated during conjugation which triggers many other important genes to do functions that are necessary for the transfer of information, such as building pili.
Vector - a plasmid that is used in lab experiments
Bibliography:
Research:
Nature News, Nature Publishing Group, www.nature.com/scitable/definition/conjugation-prokaryotes-290.
Nature News, Nature Publishing Group, www.nature.com/scitable/definition/plasmid-plasmids-28.
15paratr Follow. “Plasmids.” LinkedIn SlideShare, 22 May 2012, www.slideshare.net/15paratr/plasmids-13031139.
“Bacterial conjugation.” Wikipedia, Wikimedia Foundation, 4 Oct. 2017, en.wikipedia.org/wiki/Bacterial_conjugation.
“Biology, Eighth Edition (Raven).” Bacterial Conjugation, highered.mheducation.com/sites/9834092339/student_view0/chapter28/bacterial_conjugation.html.
“DNA Technology - Mapping a Plasmid.” Biology Junction, www.biologyjunction.com/Plasmidmapping.pdf.
“DNA/Conjugation.” DNA/Conjugation - parts.Igem.org, partsregistry.org/DNA/Conjugation.
“Joshua Lederberg.” Wikipedia, Wikimedia Foundation, 6 Nov. 2017, en.wikipedia.org/wiki/Joshua_Lederberg.
“Mechanism of Recombination, 3D animation with with basic narration :: DNA Learning Center.” DNALC Blogs, www.dnalc.org/view/15476-Mechanism-of-Recombination-3D-animation-with-with-basic-narration.html.
Monroe, Margo R. “What is a Plasmid?” Plasmids 101: What is a plasmid?, blog.addgene.org/plasmids-101-what-is-a-plasmid.
Patrick, Marcy. “So how did plasmids get their name?” Plasmids 101: A Brief History of Plasmids and an Improved eBook!, blog.addgene.org/plasmids-101-a-brief-history-of-plasmids-and-an-improved-ebook.
“Plasmid.” The Free Dictionary, Farlex, medical-dictionary.thefreedictionary.com/plasmid.
“Plasmid.” Wikipedia, Wikimedia Foundation, 2 Nov. 2017, en.wikipedia.org/wiki/Plasmid.
Boundless. “Plasmids.” Plasmids | Boundless Microbiology, courses.lumenlearning.com/boundless-microbiology/chapter/plasmids/.
“Restriction map.” Wikipedia, Wikimedia Foundation, 14 Oct. 2017, en.wikipedia.org/wiki/Restriction_map.
Media:
Picture of plasmid:
Picture of Lederberg: https://www.nobelprize.org/nobel_prizes/medicine/laureates/1958/lederberg_portrait_photo.jpg
Picture of bacteria:
https://www.quia.com/files/quia/users/lmcgee/genetics/APchapter18bacteria/conjugation.gif
Diagram of conjugation:
http://partsregistry.org/DNA/Conjugation
Video Mechanism of Recombination:
https://www.youtube.com/watch?v=8rXizmLjegI
Plasmid map:
http://www.premierbiosoft.com/images/plasmid-maps/plasmid_maps.gif
Plasmid rap video:
https://www.youtube.com/watch?v=QFfUFyUvP-w