{"id":44,"date":"2020-07-28T23:43:35","date_gmt":"2020-07-29T03:43:35","guid":{"rendered":"https:\/\/pressbooks.bccampus.ca\/kathleef\/?post_type=chapter&p=44"},"modified":"2025-06-16T16:15:36","modified_gmt":"2025-06-16T20:15:36","slug":"chapter-6-plasmids-and-cloning-basics","status":"publish","type":"chapter","link":"https:\/\/pressbooks.bccampus.ca\/kathleef\/chapter\/chapter-6-plasmids-and-cloning-basics\/","title":{"raw":"Plasmids and Cloning Basics","rendered":"Plasmids and Cloning Basics"},"content":{"raw":"

Introduction<\/h1>\r\nIn this section we will look at some qualities\/features of plasmids: the features they all have and the features that only some have. The selection of the correct plasmid for your purposes is important; otherwise work can be wasted.\r\n\r\nThen we'll consider simple restriction enzyme-based cloning: generating an insert and ligating it into your plasmid.\r\n\r\nFinally we will look at transformation of competent bacterial cells and selection of clones that have your construct. Selection of the right cells is important- depending on what you are trying to clone and how you plan to do the selection, using cells with the wrong genotype can ruin the experiment. Before purchasing competent cells, or making them yourself, ensure you know the genotype. Once you have colonies you can select individual colonies to grow up for a plasmid prep, that will give you (we hope) enough DNA for further analysis.\r\n\r\n \r\n\r\n \r\n
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Contents<\/h2>\r\n

Learning Outcomes<\/a>\r\nA. Features and use of plasmids<\/a><\/p>\r\n

A-1. Terminology<\/a>\r\nA-2. Several types of cloning vectors<\/a>\r\nA-3. Characteristics of plasmids<\/a>\r\nA-4. Types of plasmids<\/a>\r\nA-5. Plasmid names<\/a><\/p>\r\n

B. Restriction digest cloning<\/a><\/p>\r\n

B-1. Selection of Enzymes<\/a>\r\nB-2. Using PCR to add RE sites to your insert<\/a>\r\nB-3. Cloning with compatible sticky ends: molar ratio<\/a><\/p>\r\n

B3-i. TA cloning (a quick aside)<\/a><\/p>\r\n

B-4. Blunt end ligation<\/a><\/p>\r\n

C. Transformation of bacteria<\/a><\/p>\r\n

C-1. Competent cells<\/a>\r\nC-2. Transformation procedures<\/a><\/p>\r\n

C2-i. Heat shock<\/a>\r\nC2-ii. Electroporation<\/a><\/p>\r\n

C2-iii. Calculating transformation efficiency<\/a><\/p>\r\n

C-3. Selecting bacteria with the desired construct<\/a><\/p>\r\n

C3-i. Antibiotic resistance<\/a>\r\nC3-ii. Blue-white selection<\/a>\r\nC3-iii. Lac operon (review if you need it)<\/a>\r\nC3-iv. Transformation efficiency: how good are my cells?<\/a><\/p>\r\n

D. Plasmid Purification<\/a><\/p>\r\n

D-1. Alkaline lysis method<\/a><\/p>\r\n

D1-i. Plasmid conformation<\/a>\r\nD1-ii. Alkaline lysis procedure<\/a><\/p>\r\n

D-2. Sphaeroplast method (based on size)<\/a><\/p>\r\n \r\n\r\nList of lecture videos (excluding supplemental videos)<\/a>\r\n\r\n<\/div>\r\n\r\n

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<\/a>Learning Outcomes<\/h2>\r\n<\/header>\r\n
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<\/div>\r\n
\u2022Define<\/span> the terms plasmid and vector in your own words<\/div>\r\n
\u2022Distinguish<\/span> the types of plasmids covered here based on copy number, host range, and purpose<\/div>\r\n
\u2022List and describe<\/span> the common elements of cloning plasmids: what do they all have to have? What extra features do some have?<\/div>\r\n
\u2022Describe and explain<\/span> blue-white selection<\/div>\r\n
\u2022Explain or be able to draw out<\/span>\u00a0the steps of RE\/ligation-based cloning into a plasmid<\/div>\r\n
\u2022Explain<\/span> the steps of two types of plasmid prep, and the different principles of plasmid isolation<\/div>\r\n<\/div>\r\n<\/div>\r\n\r\n
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<\/a>A. Features and Use of plasmids:<\/h2>\r\n

<\/a>A-1. Terminology<\/h3>\r\nPlasmids<\/span><\/strong> are small extrachromosomal circular (usually) DNAs found in bacterial cells. Extrachromosomal means that they are separate from the bacterial chromosome. There are many different types of plasmids and a bacterium can contain different types under some circumstances. Plasmids replicate independently of the bacterial chromosome and some types of plasmids are found in many - even hundreds - of copies in each bacterial cell.\r\n\r\nMost plasmids are circular, although there are some linear plasmids. We won't be discussing these here. Plasmids perform a variety of functions in bacterial cells. You may remember the F-factor<\/span><\/strong> or the F-plasmid from introductory genetics- this is the \"fertility factor\" which allows the bacterium that contains it to form a pilus, a kind of tube that connects two bacteria, and transfer the plasmid and any genes on the plasmid to a recipient bacterium during conjugation. <\/strong>Some plasmids can actually kill the cells that take them up. Others confer resistance to antibiotics or introduce components of the restriction-modification system, which you read about in Chapter 4<\/a>. Other functions played by some types of plasmids are: allowing bacteria to tolerate heavy metals in their surroundings, protecting them from UV light, conferring the ability to parasitize plants (we'll talk about this towards the end of the course) and the ability to enter into symbiosis with plants.\u00a0 In this last case, legumes (peas, beans, peanuts, soybeans etc) form nodules which house the bacteria and in return the bacteria fix nitrogen from the atmosphere to allow the plants to grow better. It seems to be a mutually beneficial arrangement.<\/span><\/span>\r\n\r\nWhat is interesting about plasmids is that they can undergo horizontal transfer<\/strong><\/span>. This means that they are not passed only between bacteria of the same species. Instead they can be passed among many different species of bacteria. Horizontal transfer results in genetic changes that were not inherited through reproduction (which is called vertical transfer<\/span><\/strong>). Bacterial strains that have acquired multiple drug resistance most likely did so through acquiring plasmids that contain the genes for resistance to antibiotics from other species of bacteria.\r\n\r\nPlasmids are a type of cloning vector.<\/strong><\/span> A vector is used to introduce DNA into cells. There are many types of vector, which is a more general term, and plasmids (the more specific term) are one type.\r\n\r\n \r\n\r\n \r\n\r\n
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<\/a>A-2. Several types of cloning vectors<\/h3>\r\nAlthough we are focused here on plasmids, you should\u00a0 know that many other types of vectors exist for cloning purposes. We'll briefly describe these. If you work in a lab that uses these, there will be an opportunity to learn much more; this serves just as a brief introduction. Different types of vectors are used for cloning different sized inserts.\r\n\r\nPlasmids are used in bacteria and can hold inserts up to about 15 kb, though often the inserts are smaller than this.\r\n\r\nLambda phage <\/strong>i<\/span><\/span>s<\/span> a virus, and it can be used to transfer inserts of up to about 25 kb into recipient bacterial cells. The viral particles contain the insert in place of the genes needed to replicate more viruses. Thus although the viruses can infect the bacterial cells, no new viruses are formed in the hosts. In addition to being able to transfer larger insert DNAs into recipient cells, viruses have the advantage of being more efficient at infecting cells than the transformation process we use with plasmids (discussed in part C)\r\n\r\nCosmids<\/strong><\/span> have features of lambda phage and plasmids- they use the efficiency of viral infection to transfer the DNA into recipient bacterial cells but once the DNA is inside the bacteria, we can work with the DNA as a plasmid. Cosmids can transfer up to 45 kb into the recipient bacterial cells.\r\n\r\nArtificial chromosomes<\/span> <\/strong>of various types\u00a0 (P1 artificial chromosomes and bacterial artificial chromosomes, or BACs) are used to transfer even larger amounts of DNA into recipient cells, mostly bacteria. \u00a0In the case of yeast artificial chromosomes (YACs) which can transfer 250 kb to 2 Mb of sequence, the recipient cells are yeasts. Yeasts are extremely useful organisms because they are eukaryotes, but you can culture them in large numbers like bacteria. Artificial chromosomes have all the components needed for a real chromosome: an origin of replication, and if the chromosome is a YAC, telomere and centromere sequences that allow the chromosome to survive in the cell and segregate properly during cell division. The rest is the inserted DNA.\r\n\r\n \r\n\r\n[h5p id=\"19\"]\r\n\r\n
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<\/a>A-3. Characteristics of plasmids<\/h3>\r\nAll plasmids- that we use for cloning - have the following features:\r\n