Plasmids and Plasmid Maps BEGINS

Mr Badbacte Vs Mr Goodbacter. HI there boys and girls. I’m Gumby and I will be teaching you all about this thing called the plasmid. We use it a lot in biotechnology so it is really important.

The reason the plasmids are so important is that they are used by bacteria in a type of exchange of genetic information. Its a little like sex for bacteria –but its called conjugation. The method is also called HORIZONTAL GENE TRANSFER. Hey when can my wife come for a conjugal visit? pili Hi there partner in crime. What’s goin’ on here?

You can notice in this bacterial cell that the plasmids are in blue and the bacterial genome is in red. The bacterial genome is circular and it is a lot larger than the plasmid. It is needed by the bacteria to perform all the functions necessary for it to survive. The plasmids are like a little bonus. They can transfer little pieces of information like antibiotic resistance for example. Because of the smaller size of plasmids, they are easier to move into bacterial cells

This is what it’s all about. You see the plasmid is used as a vector (method of transport) for cloned genes. You can see this plasmid has been cut and a foreign gene inserted in. The bacteria with this plasmid are now the clones and they can manufacture a gene product.

Hey kids. Say hello to my good friend Mr. Goodbacter. He is a transformed bacteria by genetic engineering. You can see the little plasmid in his chin. Say hello! Thanks for the welcome. You know, I’m really excited about producing great products to help humanity. So far I have clones that produce valuable hormonal products like insulin and growth hormone. Scientists are working on many more as we speak. You lousy traitor. You’re not supposed to be helping humans. You should be giving them infections and stuff.

Say, who’s your friend Mr. Goodbacter ? His name is Mr. Badbacter and he tries really hard to give all us bacteria a bad reputation. He thinks that we bacteria should all cause disease, but I know that the vast majority of bacteria do not do that. Most bacteria are helpful for the environment specifically decompsition. Did you know that some bacteria can even help clean up oil spills? I’m helpful in a very new scientific way, thanks to our brainy scientists. Hey Goodbacter. What did these “scientists” ever do for you?

So these plasmids offer a lot of possibilities for the bacteria to produce things we never thought possible. Scientists use us because we are easy to grow and we multiply rapidly. This makes it easy for them to experiment and determine if a plasmid gets into a bacteria. For instance, if they are not sure that the bacteria obtained a particular plasmid, they can check by placing us all on an antibiotic plate. Only those with the plasmid will survive because the plasmid transfers antibiotic resistance.

Plasmids can be cut open and new genes can be inserted. In order to do this scientists are interested in where the cuts are located on the plasmid. It is important, for instance, that when you use a restriction enzyme (ex EcoRI) it does not cut through something important already on the plasmid. For instance, you would not want the EcoRI to cut in the middle of the antibiotic resistance gene, because that would ruin the bacteria’s ability to produce the resistance to the antibiotic. In order to figure out where all these genes are and where we will cut with a specific restriction enzyme, we need something called a PLASMID MAP

To start to understand how to map a plasmid, lets take an example. Lets say that a particular plasmid is only cut once by the restriction enzyme EcoRI= scissors This all happens on a petri dish, so we cannot see it. We need to use electrophoresis to visualize what has happened. Since the plasmid is a circle, if it has been cut once, it forms one length of DNA. Run it on the get to see. 4500bp 4000bp 3500bp 2000bp 500bp 1500bp 2500bp 3000bp Reference sample Plasmd sample Gel electrophoresis Sample is 4000bp long

To start to understand how to map a plasmid, lets take an example. Lets say that a particular plasmid is only cut once by the restriction enzyme EcoRI= scissors This all happens on a petri dish, so we cannot see it. We need to use electrophoresis to visualize what has happened. Since the plasmid is a circle, if it has been cut once, it forms one length of DNA. Run it on the get to see. 4500bp 4000bp 3500bp 2000bp 500bp 1500bp 2500bp 3000bp Reference sample Plasmd sample Gel electrophoresis Sample is 4000bp long Plasmid A 4000 bp total length 4000 bp

So in this instance the plasmid is 4000 bp (nucleotide base pairs) long. If we try to cut the plasmid with another restriction endonuclease it will not cut in the same spot. We will represent this other restriction enzyme with a different pair of scissors. Hind III Reference sample Plasmd sample From the gel it is apparent that there are two pieces of DNA and thus two cuts of the plasmid. Comparing the lines to the reference sample, one length is 1000bp, the other 3000bp. Plasmid A 4000 bp total length Note how the number of base pairs remains the same = bp 3000 bp

This time I’ll let you figure out the number of cuts and the size of the plasmid. We will use a new plasmid, Plasmid B, this time. There are two cuts on this plasmid with restriction enzyme EcoRI. The two DNA lengths are 1000 bp and 1500 bp. This means that the total length of the plasmid is 2500 bp. A diagram is provided above. EcoRI Plasmid B 2500 bp total What can you tell from the gel? 1000 bp 1500 bp

This time we’ll take a little short cut. Instead of having you look at the gel, we will just get the information off a chart. EcoRi (base pairs) 1800 Results of restriction enzyme digestion of plasmid C Plasmid C 1800 bp total Bam HIi (base pairs) EcoRI BamHI Plasmid C 1800 bp total Notice these cut at different locations 600 bp 1200 bp 1800 bp

Now for the next one we will do a double digest utilizing two different restriction endonucleases. EcoRI and BamHI We will still use plasmid C. Hold on tight! EcoRIBamHIEcoRI + BamHI 1800 bp1200 bp200 bp 600 bp 1000 bp These add together to make the 1800 total This is a double digest. The plasmid is placed in a container with both of the restriction enzymes Position the EcoRI 1800/0 start Plasmid C 1800 bp total Determine which Bam is spliced by the EcoRI. Here 1200 is cut to 200 and 1000 in the double digest Position the 1200 Bam so that the EcoRI cuts it into two pieces, one 1000bp(dark green) and the other 200bp(light green) bp 600

The reverse orientation is also correct in this plasmid map Finally after colour coding the splices we make the final map You only have to choose one. They are both correct 1800/0 bp EcoRi 200bp BamHI 800 bp BamHI 1000bp BamHI 1600bp BamHI This is the direction of the number system.

You will get some experience working on these plasmid maps from the Plasmid map worksheets you are to complete. When completing your plasmid map activity, you are to include one working diagram with the colour code and base pair numbers on it. You also will include your final plasmid map showing restriction endonuclease cuts in the appropriate locations. 1800/0 bp EcoRi 200bp BamHI 800 bp BamHI EcoRI must be at top

The worksheets have been made a little easier for mapping by utilizing a plasmid of 1200 bp length. This can be compared to a clock to make interpretation easier. EcoRI always at top1200/0 EcoRIBamHIEcoRI + BamHI 1200 bp800 bp300 bp 400 bp 500 bp 300 bp BamHI 700 bp BamHI

Here is an example of a real plasmid map. Gee, Mr. Goodbacter. It sure is complicated.

It has to be. A lot of these plasmids have control sequences to control the inserted genome. Antibiotic resistant genes also have been placed already to make the plasmid easy to identify. Wow. That must have taken a lot of work to design that plasmid map. It looks like they have inserted quite a few foreign genes into it alreadyl

And here’s one last example showing the more complicated plasmid maps and the associated gel Biotech sure is interesting.

PLASMID AND PLASMID MAPS END I’m not licked yet. Maybe I can convince some of these transformed bacteria to go to the Dark Side. You know, with the power of genetic manipulation, maybe they could be even more dangerous.