Editing Plasmid (section)

== Study of plasmids ==

=== Plasmid DNA extraction ===
Plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for [[cloning#Molecular cloning|molecular cloning]], plasmids often need to be isolated.

There are several methods to [[plasmid preparation|isolate plasmid DNA]] from bacteria, ranging from the plasmid extraction kits ([[Plasmid preparation#Preparations by size|miniprep to the maxiprep or bulkprep]]), [[alkaline lysis]], enzymatic lysis, and mechanical lysis  .<ref name="Molecular cloning"/> The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by [[restriction digest]] and for some cloning techniques.

In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. In essence, this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several hundred micrograms) of very pure plasmid DNA.

Many commercial kits have been created to perform plasmid extraction at various scales, purity, and levels of automation.

=== Conformations ===
Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during [[agarose gel electrophoresis|electrophoresis]]. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
* ''[[Nick (DNA)|Nicked open-circular]]'' DNA has one strand cut.
* ''Relaxed circular'' DNA is fully intact with both strands uncut but has been enzymatically ''relaxed'' (supercoils removed). This can be modeled by letting a twisted extension cord unwind and relax and then plugging it into itself.
* ''Linear'' DNA has free ends, either because both strands have been cut or because the DNA was linear ''in vivo''. This can be modeled with an electrical extension cord that is not plugged into itself.
* ''[[DNA supercoil|Supercoiled]]'' (or ''covalently closed-circular'') DNA is fully intact with both strands uncut, and with an integral twist, resulting in a compact form. This can be modeled by twisting an [[extension cord]] and then plugging it into itself.
* ''Supercoiled [[denaturation (biochemistry)|denatured]]'' DNA is similar to ''supercoiled DNA'', but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continuously increasing yet different rates. Thus, the resolution of a gel decreases with increased voltage.

At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length.  Large linear fragments (over 20 kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'respirate', with the bulk of the molecule following the leading end through the gel matrix. [[Restriction digest]]s are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after [[gel electrophoresis]]. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.

Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.

=== Software for bioinformatics and design ===
{{main|List of genetic engineering software}}

The use of plasmids as a technique in [[molecular biology]] is supported by [[bioinformatics]] [[software]]. These programs record the [[DNA]] sequence of plasmid vectors, help to predict cut sites of [[restriction enzymes]], and to plan manipulations. Examples of software packages that handle plasmid maps are ApE, [[Clone manager|Clone Manager]], GeneConstructionKit, Geneious, [[Genome Compiler]], LabGenius, Lasergene, [[MacVector]], pDraw32, Serial Cloner, [[UGENE]], VectorFriends, [[Vector NTI]], and WebDSV. These pieces of software help conduct entire experiments in silico before doing wet experiments.<ref>{{cite web |url=http://vimeo.com/57923864 |title=Vector NTI feedback video |work= The DNA Lab }}</ref>

=== Plasmid collections ===
Many plasmids have been created over the years and researchers have given out plasmids to plasmid databases such as the non-profit organisations [https://www.addgene.org Addgene] and [https://bccm.belspo.be/about-GeneCorner BCCM/GeneCorner]. One can find and request plasmids from those databases for research.
Researchers also often upload plasmid sequences to the [https://www.ncbi.nlm.nih.gov/nuccore/ NCBI database], from which sequences of specific plasmids can be retrieved. There have been multiple efforts to create curated and quality controlled databases from these uploaded sequences; an early example is by Orlek ''et al'',<ref>{{cite journal |last1=Orlek |first1=Alex |last2=Phan |first2=Hang |last3=Sheppard |first3=Anna E. |last4=Doumith |first4=Michel |last5=Ellington |first5=Matthew |last6=Peto |first6=Tim |last7=Crook |first7=Derrick |last8=Walker |first8=A. Sarah |last9=Woodford |first9=Neil |last10=Anjum |first10=Muna F. |last11=Stoesser |first11=Nicole |title=A curated dataset of complete Enterobacteriaceae plasmids compiled from the NCBI nucleotide database |journal=Data in Brief |date=June 2017 |volume=12 |pages=423–426 |doi=10.1016/j.dib.2017.04.024 |pmc=5426034 |pmid=28516137 |bibcode=2017DIB....12..423O }}</ref> which limited itself to ''[[Enterobacteriaceae]]'' plasmids, while COMPASS also encompassed plasmids from other bacteria. More recently, PLSDB<ref>{{cite journal |last1=Schmartz |first1=Georges P |last2=Hartung |first2=Anna |last3=Hirsch |first3=Pascal |last4=Kern |first4=Fabian |last5=Fehlmann |first5=Tobias |last6=Müller |first6=Rolf |last7=Keller |first7=Andreas |title=PLSDB: advancing a comprehensive database of bacterial plasmids |journal=Nucleic Acids Research |date=7 January 2022 |volume=50 |issue=D1 |pages=D273–D278 |doi=10.1093/nar/gkab1111 |pmc=8728149 |pmid=34850116 }}</ref> was made as a more up to date curated database of NCBI plasmids, and as of 2024 contains over 72,000 entries.<ref>{{cite journal |last1=Molano |first1=Leidy-Alejandra G |last2=Hirsch |first2=Pascal |last3=Hannig |first3=Matthias |last4=Müller |first4=Rolf |last5=Keller |first5=Andreas |title=The PLSDB 2025 update: enhanced annotations and improved functionality for comprehensive plasmid research |journal=Nucleic Acids Research |date=6 January 2025 |volume=53 |issue=D1 |pages=D189–D196 |doi=10.1093/nar/gkae1095 |pmc=11701622 |pmid=39565221 }}</ref> A similar database is pATLAS, which additionally includes visual analytics tools to show relationships between plasmids.<ref>{{cite journal |last1=Jesus |first1=Tiago F |last2=Ribeiro-Gonçalves |first2=Bruno |last3=Silva |first3=Diogo N |last4=Bortolaia |first4=Valeria |last5=Ramirez |first5=Mário |last6=Carriço |first6=João A |title=Plasmid ATLAS: plasmid visual analytics and identification in high-throughput sequencing data |journal=Nucleic Acids Research |date=8 January 2019 |volume=47 |issue=D1 |pages=D188–D194 |doi=10.1093/nar/gky1073 |pmc=6323984 |pmid=30395323 }}</ref> The largest plasmid database made from publicly available data is IMG/PR, which not only contains full plasmid sequences retrieved from NCBI, but novel plasmid genomes found from [[Metagenomics|metagenomes]] and metatranscriptomes.<ref>{{cite journal |last1=Camargo |first1=Antonio Pedro |last2=Call |first2=Lee |last3=Roux |first3=Simon |last4=Nayfach |first4=Stephen |last5=Huntemann |first5=Marcel |last6=Palaniappan |first6=Krishnaveni |last7=Ratner |first7=Anna |last8=Chu |first8=Ken |last9=Mukherjeep |first9=Supratim |last10=Reddy |first10=T B K |last11=Chen |first11=I-Min A |last12=Ivanova |first12=Natalia N |last13=Eloe-Fadrosh |first13=Emiley A |last14=Woyke |first14=Tanja |last15=Baltrus |first15=David A |last16=Castañeda-Barba |first16=Salvador |last17=de la Cruz |first17=Fernando |last18=Funnell |first18=Barbara E |last19=Hall |first19=James P J |last20=Mukhopadhyay |first20=Aindrila |last21=Rocha |first21=Eduardo P C |last22=Stalder |first22=Thibault |last23=Top |first23=Eva |last24=Kyrpides |first24=Nikos C |title=IMG/PR: a database of plasmids from genomes and metagenomes with rich annotations and metadata |journal=Nucleic Acids Research |date=5 January 2024 |volume=52 |issue=D1 |pages=D164–D173 |doi=10.1093/nar/gkad964 |pmc=10767988 |pmid=37930866 }}</ref>

Other datasets have been created by sequencing and computing plasmid genomes from pre-existing bacterial collections, e.g. the NORM collection<ref>{{Cite journal |last1=Gladstone |first1=Rebecca A. |last2=McNally |first2=Alan |last3=Pöntinen |first3=Anna K. |last4=Tonkin-Hill |first4=Gerry |last5=Lees |first5=John A. |last6=Skytén |first6=Kusti |last7=Cléon |first7=François |last8=Christensen |first8=Martin O. K. |last9=Haldorsen |first9=Bjørg C. |last10=Bye |first10=Kristina K. |last11=Gammelsrud |first11=Karianne W. |last12=Hjetland |first12=Reidar |last13=Kümmel |first13=Angela |last14=Larsen |first14=Hege E. |last15=Lindemann |first15=Paul Christoffer |date=2021-07-01 |title=Emergence and dissemination of antimicrobial resistance in Escherichia coli causing bloodstream infections in Norway in 2002–17: a nationwide, longitudinal, microbial population genomic study |journal=The Lancet Microbe |language=English |volume=2 |issue=7 |pages=e331–e341 |doi=10.1016/S2666-5247(21)00031-8 |pmc=7614948 |pmid=35544167}}</ref><ref>{{cite report |type=Preprint |doi=10.1101/2023.10.14.562336 |title=Plasmid-driven strategies for clone success in ''Escherichia coli'' |date=2024 |last1=Arredondo-Alonso |first1=Sergio |last2=Pöntinen |first2=Anna K. |last3=Gama |first3=João Alves |last4=Gladstone |first4=Rebecca A. |last5=Harms |first5=Klaus |last6=Tonkin-Hill |first6=Gerry |last7=Thorpe |first7=Harry A. |last8=Simonsen |first8=Gunnar S. |last9=Samuelsen |first9=Ørjan |last10=Johnsen |first10=Pål J. |last11=Corander |first11=Jukka }}</ref> and the Murray Collection.<ref>{{cite journal |last1=Baker |first1=Kate S. |last2=Burnett |first2=Edward |last3=McGregor |first3=Hannah |last4=Deheer-Graham |first4=Ana |last5=Boinett |first5=Christine |last6=Langridge |first6=Gemma C. |last7=Wailan |first7=Alexander M. |last8=Cain |first8=Amy K. |last9=Thomson |first9=Nicholas R. |last10=Russell |first10=Julie E. |last11=Parkhill |first11=Julian |title=The Murray collection of pre-antibiotic era Enterobacteriacae: a unique research resource |journal=Genome Medicine |date=December 2015 |volume=7 |issue=1 |page=97 |doi=10.1186/s13073-015-0222-7 |doi-broken-date=10 March 2025 |doi-access=free |pmc=4584482 |pmid=26411565 }}</ref><ref>{{cite report |type=Preprint |doi=10.1101/2024.09.03.610986 |title=Pre and Post antibiotic epoch: Insights into the historical spread of antimicrobial resistance |date=2024 |last1=Cazares |first1=Adrian |last2=Figueroa |first2=Wendy |last3=Cazares |first3=Daniel |last4=Lima |first4=Leandro |last5=Turnbull |first5=Jake D. |last6=McGregor |first6=Hannah |last7=Dicks |first7=Jo |last8=Alexander |first8=Sarah |last9=Iqbal |first9=Zamin |last10=Thomson |first10=Nicholas R. }}</ref>