Difference between revisions of "Help:Plasmid backbones/Features"

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[[Help:Plasmid backbones|< Back to Plasmid backbone Help]]
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https://static.igem.org/mediawiki/parts/4/45/Plasmidfeatures.png
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==Antibiotic Resistance==
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By incorporating an antibiotic-resistance gene on a plasmid, engineered-cells are able to be selected for using the appropriate antibiotic. The most common resistances are ampicillin ("Amp"), kanomycin ("Kan"), tetracycline ("Tet") and chloramphenicol ("Chlor").  For more information on which plasmids carry these resistances, click the [[Help:Plasmids/Nomenclature |nomenclature]] documentation section.   
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==Copy Number==
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[[Image:BioBrickvectorbackbone.png|right]]
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A plasmid's 'copy number' refers to how many times the plasmid self-replicates within a cell (via the origin of replication or ORI) and thus how many copies are present. This can play a significant role in intracellular systems, particularly BioBricked synthetic devices. Large DNA inserts can greatly reduce the copy number of a plasmid, despite the fact that they may originally be high-copy.
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Most Biobricks are ''"high copy"'' type plasmids and are well-suited for high yield plasmid purification.  These plasmids can reach large numbers by being replicated using host enzymes (source: [http://www.biochem.wisc.edu/courses/biochem651_f05/Lectures/08Plasmids/Theory_plasmids/copynumber.html  University of Wisc. Madison])
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<font size="4">'''Plasmid backbones tend to have three key features'''</font>
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==Origin (of replication)==
 
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The origin of replication is a binding sequence where replication proteins which nick, unwind, and replicate plasmid DNA independently of the cellular host.
 
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Many biobrick plasmids have the following type of origin of replication:
 
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*<b>pmB1</b> - This origin has been isolated from one of the first plasmids taken from a human body.  It is a "relaxed" plasmid, meaning that it allows high copy amounts within a cell
 
  
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Note: This origin is not to be confused with the "origin of ''transfer''", the site used in conjugal rolling circle replication transfer.
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<font size="4">1. Replication origin</font>
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{{:Help:Plasmid backbones/Features/Replication_origin}}
  
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==Size==
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Thus, you often [[Help:Plasmid backbones/Choosing the right plasmid|choose your plasmid backbone replication origin]] based on your application.
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The larger your plasmid is, the more difficult it will be for the cell to produce large quantities of it.  Since the size of a plasmid can be determined by both the size of the bare [[Help:Plasmids/Construction_Plasmids#Plasmid (backbone)|plasmid backbone]] and the your construct inside of it.  Thus it is generally a good idea to use plasmids of a small size.
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<font size="4">2.  Antibiotic resistance marker</font>
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{{:Help:Plasmid backbones/Features/Antibiotic_resistance_marker}}
  
  
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==Links and References==
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<font size="4">3. Cloning site</font>
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*[http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/cloningvectors.html "Cloning Vectors and Genetic Engineering"] - from educators at Rutgers University
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*[http://www1.qiagen.com/Plasmid/BacterialCultures.aspx?#tab2 "Plasmid Applications"] - Qiagen's guide
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The '''cloning site''' is the location on the plasmid backbone where BioBrick parts are inserted. Most plasmid backbones in microbiology have "multiple cloning site" because the plasmid backbone has several restriction sites in a row allowing you to choose where you insert your DNA fragment and which enzymes you use. In contrast, BioBrick plasmid backbones have a BioBrick cloning site consisting of a BioBrick prefix (including enzyme recognition sites for EcoRI and XbaI) and a BioBrick suffix (including enzyme recognition sites for SpeI and PstI). All BioBrick parts are inserted at the BioBrick cloning site between the BioBrick prefix and BioBrick suffix. The prefix sequence occurs ''before'' the BioBrick part, and the suffix sequence occurs ''after'' the BioBrick part.
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Latest revision as of 18:51, 10 December 2008

< Back to Plasmid backbone Help

BioBrickvectorbackbone.png

Plasmid backbones tend to have three key features


1. Replication origin

The genetic element responsible for the replication of plasmids during cell growth and division is called a replication origin (also "origin of replication" or simply "origin"). There are several different replication origins and they differ in their plasmid copy number per cell (how many molecules of the plasmid are maintained in the cell), mechanism of copy number control, cell-to-cell copy number variation, and even the degree of coiling of the physical DNA. Thus, BioBrick® parts, devices and systems can operate very differently from one plasmid backbone to another.

Thus, you often choose your plasmid backbone replication origin based on your application.


2. Antibiotic resistance marker

At the most basic level, function of the antibiotic resistance marker is to allow the cell to grow even in the presence of a particular antibiotic. Plasmid backbones include antibiotic resistance markers because the markers allow you to select for cells that contain your plasmid. When E. coli cells grow and divide, plasmids can inadvertently be lost from the cell. In some cases, cells without a plasmid can potentially grow faster than cells with the plasmid which means that cell cultures can quickly become dominated by plasmid-free cells. Since most BioBrick® parts are maintained on plasmid backbones, plasmid loss is problematic. To help avoid these problems, every plasmid backbone includes an antibiotic resistance marker. Thus, cells which don't have a copy of the plasmid are killed by antibiotic present. Common antibiotic resistance markers in BioBrick® plasmid backbones confer resistance to ampicillin ("Amp" or A), kanamycin ("Kan" or K), chloramphenicol ("Cm" or C) and tetracycline ("Tet" or T).


3. Cloning site

The cloning site is the location on the plasmid backbone where BioBrick parts are inserted. Most plasmid backbones in microbiology have "multiple cloning site" because the plasmid backbone has several restriction sites in a row allowing you to choose where you insert your DNA fragment and which enzymes you use. In contrast, BioBrick plasmid backbones have a BioBrick cloning site consisting of a BioBrick prefix (including enzyme recognition sites for EcoRI and XbaI) and a BioBrick suffix (including enzyme recognition sites for SpeI and PstI). All BioBrick parts are inserted at the BioBrick cloning site between the BioBrick prefix and BioBrick suffix. The prefix sequence occurs before the BioBrick part, and the suffix sequence occurs after the BioBrick part.