Editing Plasmid (section)

==Vectors==
{{Further|Vector (molecular biology)}}
Artificially constructed plasmids may be used as [[vector (molecular biology)|vectors]] in [[genetic engineering]].  These plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to clone and amplify (make many copies of) or [[gene expression|express]] particular genes.<ref name="Molecular cloning">{{cite book | vauthors = Russell DW, Sambrook J |title=Molecular cloning: a laboratory manual |publisher=Cold Spring Harbor Laboratory |location=Cold Spring Harbor, NY |year=2001 }}</ref> A wide variety of plasmids are commercially available for such uses. The gene to be replicated is normally inserted into a plasmid that typically contains a number of features for their use.  These include a gene that confers resistance to particular antibiotics ([[ampicillin]] is most frequently used for bacterial strains), an [[origin of replication]] to allow the bacterial cells to replicate the plasmid DNA, and a suitable site for cloning (referred to as a [[multiple cloning site]]).

DNA structural instability can be defined as a series of spontaneous events that culminate in an unforeseen rearrangement, loss, or gain of genetic material. Such events are frequently triggered by the transposition of mobile elements or by the presence of unstable elements such as non-canonical (non-B) structures. Accessory regions pertaining to the bacterial backbone may engage in a wide range of structural instability phenomena. Well-known catalysts of [[genetic instability]] include direct, inverted, and tandem repeats, which are known to be conspicuous in a large number of commercially available cloning and expression vectors.<ref>{{cite journal | vauthors = Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA | title = Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events | journal = Applied Microbiology and Biotechnology | volume = 87 | issue = 6 | pages = 2157–2167 | date = August 2010 | pmid = 20496146 | doi = 10.1007/s00253-010-2671-7 | s2cid = 19780633 | doi-access = free }}</ref> Insertion sequences can also severely impact plasmid function and yield, by leading to [[deletion (genetics)|deletion]]s and rearrangements, activation, [[down-regulation]] or inactivation of neighboring [[gene expression]].<ref>{{cite journal | vauthors = Gonçalves GA, Oliveira PH, Gomes AG, Prather KL, Lewis LA, Prazeres DM, Monteiro GA | title = Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters | journal = Applied Microbiology and Biotechnology | volume = 98 | issue = 15 | pages = 6609–6619 | date = August 2014 | pmid = 24769900 | doi = 10.1007/s00253-014-5695-6 | hdl-access = free | s2cid = 9826684 | hdl = 1721.1/104375 }}</ref> Therefore, the reduction or complete elimination of extraneous [[noncoding DNA|noncoding]] backbone sequences would pointedly reduce the propensity for such events to take place, and consequently, the overall recombinogenic potential of the plasmid.<ref>{{cite journal | vauthors = Oliveira PH, Mairhofer J | title = Marker-free plasmids for biotechnological applications - implications and perspectives | journal = Trends in Biotechnology | volume = 31 | issue = 9 | pages = 539–547 | date = September 2013 | pmid = 23830144 | doi = 10.1016/j.tibtech.2013.06.001 }}</ref><ref>{{cite journal | vauthors = Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA | title = Structural instability of plasmid biopharmaceuticals: challenges and implications | journal = Trends in Biotechnology | volume = 27 | issue = 9 | pages = 503–511 | date = September 2009 | pmid = 19656584 | doi = 10.1016/j.tibtech.2009.06.004 }}</ref>

[[File:pBR322.svg|thumb|A schematic representation of the [[pBR322]] plasmid, one of the first plasmids to be used widely as a [[cloning vector]].  Shown on the plasmid diagram are the genes encoded (''amp'' and ''tet'' for [[ampicillin]] and [[tetracycline]] resistance respectively), its origin of replication (''ori''), and various [[restriction site]]s (indicated in blue).]]

===Cloning===
{{main|Cloning vector}}
Plasmids are the most-commonly used bacterial cloning vectors.<ref name="uldis">{{cite book | vauthors = Geoghegan T | chapter = Molecular Applications | chapter-url=https://books.google.com/books?id=1wyf7pbR5z4C&pg=PA248 |title=Modern Microbial Genetics | veditors = Streips UN, Yasbin RE |publisher=Wiley-Blackwell |edition= 2nd |year= 2002 |isbn= 978-0471386650 |page=248 }}</ref> These cloning vectors contain a site that allows DNA fragments to be inserted, for example a [[multiple cloning site]] or polylinker which has several commonly used [[restriction sites]] to which DNA fragments may be [[Ligation (molecular biology)|ligated]]. After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called [[transformation (genetics)|transformation]]. These plasmids contain a [[selectable marker]], usually an antibiotic resistance gene, which confers on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics.  The cells after transformation are exposed to the selective media, and only cells containing the plasmid may survive.  In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA.  The vector may also contain other [[marker gene]]s or [[reporter gene]]s to facilitate selection of plasmids with cloned inserts.  Bacteria containing the plasmid can then be grown in large amounts, harvested, and the plasmid of interest may then be isolated using various methods of [[plasmid preparation]].

A plasmid cloning vector is typically used to clone DNA fragments of up to 15 [[base pair|kbp]].<ref>{{cite book | vauthors = Preston A |chapter=Chapter 2 – Choosing a Cloning Vector |pages=19–26 |chapter-url=https://books.google.com/books?id=r6QC0hTwsrwC&pg=PA19  | veditors = Casali N, Preston A  |title=E. Coli Plasmid Vectors: Methods and Applications|series=Methods in Molecular Biology | volume = 235 |publisher=Humana Press |year= 2003  |isbn=978-1-58829-151-6}}</ref> To clone longer lengths of DNA, [[lambda phage]] with lysogeny genes deleted, [[cosmid]]s, [[bacterial artificial chromosome]]s, or [[yeast artificial chromosome]]s are used.

=== Suicide Vectors (plasmids) ===
Suicide vectors are plasmids that are unable to replicate in the host cell and therefore have to integrate in the chromosome or disappear.<ref>{{cite book |doi=10.1016/B978-0-12-385075-1.00024-X |chapter=Synthetic Biology in Cyanobacteria |title=Synthetic Biology, Part A |series=Methods in Enzymology |date=2011 |last1=Heidorn |first1=Thorsten |last2=Camsund |first2=Daniel |last3=Huang |first3=Hsin-Ho |last4=Lindberg |first4=Pia |last5=Oliveira |first5=Paulo |last6=Stensjö |first6=Karin |last7=Lindblad |first7=Peter |volume=497 |pages=539–579 |pmid=21601103 |isbn=978-0-12-385075-1 |quote=Integrative plasmids are in most cases suicide vectors, that is, vectors that are unable to replicate in the destination host and therefore must either integrate or disappear, and hence, any plasmid that can be efficiently transferred into the recipient may be used. }}</ref> One example of these vectors are pMQ30 plasmid. This plasmid has SacB gene from ''Bacillus subtilis'' which can be  induced by sucrose and it'll be lethal when expressed in Gram-negative bacteria.<ref>{{cite journal |last1=Quandt |first1=Jürgen |last2=Hynes |first2=Michael F. |title=Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria |journal=Gene |date=May 1993 |volume=127 |issue=1 |pages=15–21 |doi=10.1016/0378-1119(93)90611-6 |pmid=8486283 }}</ref> The benefit of this system( two-step success monitoring ) shows when the experiment design needs a target gene to be integrated into the chromosome of the bacterial host. In the first step after transforming the host cells with the plasmid, a media with specific antibiotic could be used to select for bacteria that contain the plasmid. The second step makes sure that only the bacteria with integrated plasmid would survive. Since the plasmid contain the SacB gene that will induce toxicity in presence of sucrose, only the bacteria would survive and grow that has the plasmid integrated in their chromosome.

===Protein Production===
{{main|Expression vector}}
[[File:Human insulin 100IU-ml vial white background.jpg|thumb|Insulin]]
Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing the protein, for example, utilizing the rapid reproduction of E.coli with a plasmid containing the [[insulin]] gene leads to a large production of insulin.<ref>{{Cite journal |last1=Goeddel |first1=D V |last2=Kleid |first2=D G |last3=Bolivar |first3=F |last4=Heyneker |first4=H L |last5=Yansura |first5=D G |last6=Crea |first6=R |last7=Hirose |first7=T |last8=Kraszewski |first8=A |last9=Itakura |first9=K |last10=Riggs |first10=A D |date=January 1979 |title=Expression in Escherichia coli of chemically synthesized genes for human insulin. |journal=Proceedings of the National Academy of Sciences |volume=76 |issue=1 |pages=106–110 |doi=10.1073/pnas.76.1.106 |doi-access=free |pmc=382885 |pmid=85300 |bibcode=1979PNAS...76..106G }}</ref><ref>{{cite journal |last1=Govender |first1=Kamini |last2=Naicker |first2=Tricia |last3=Lin |first3=Johnson |last4=Baijnath |first4=Sooraj |last5=Chuturgoon |first5=Anil Amichund |last6=Abdul |first6=Naeem Sheik |last7=Docrat |first7=Taskeen |last8=Kruger |first8=Hendrik Gerhardus |last9=Govender |first9=Thavendran |title=A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli) |journal=AMB Express |date=December 2020 |volume=10 |issue=1 |page=43 |doi=10.1186/s13568-020-00969-w |doi-access=free |pmc=7062966 |pmid=32152803 }}</ref><ref>{{Cite journal |last1=Zieliński |first1=Marcin |last2=Romanik-Chruścielewska |first2=Agnieszka |last3=Mikiewicz |first3=Diana |last4=Łukasiewicz |first4=Natalia |last5=Sokołowska |first5=Iwona |last6=Antosik |first6=Jarosław |last7=Sobolewska-Ruta |first7=Agnieszka |last8=Bierczyńska-Krzysik |first8=Anna |last9=Zaleski |first9=Piotr |last10=Płucienniczak |first10=Andrzej |date=2019-05-01 |title=Expression and purification of recombinant human insulin from E. coli 20 strain |journal=Protein Expression and Purification |volume=157 |pages=63–69 |doi=10.1016/j.pep.2019.02.002 |pmid=30735706 |doi-access=free }}</ref>

===Gene therapy===
{{main|Vectors in gene therapy}}
Plasmids may also be used for gene transfer as a potential treatment in [[gene therapy]] so that it may express the protein that is lacking in the cells.  Some forms of [[gene therapy]] require the insertion of therapeutic [[gene]]s at pre-selected [[chromosome|chromosomal]] target sites within the human [[genome]]. Plasmid vectors are one of many approaches that could be used for this purpose. [[Zinc finger nuclease]]s (ZFNs) offer a way to cause a site-specific [[double-strand break]] to the DNA genome and cause [[homologous recombination]]. Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that [[cell damage]], cancer-causing mutations, or an [[immune response]] is avoided.<ref name= Kandavelou>{{cite book |vauthors=Kandavelou K, Chandrasegaran S |year=2008|chapter=Plasmids for Gene Therapy|title=Plasmids: Current Research and Future Trends|publisher=Caister Academic Press|isbn= 978-1-904455-35-6}}</ref>

===Disease models===
Plasmids were historically used to genetically engineer the embryonic stem cells of rats to create rat genetic disease models. The limited efficiency of plasmid-based techniques precluded their use in the creation of more accurate human cell models. However, developments in [[adeno-associated virus]] recombination techniques, and [[zinc finger nucleases]], have enabled the creation of a new generation of [[isogenic human disease models]].

=== Biosynthetic Gene Cluster (BGC) ===
Plasmids assist in transporting [[biosynthetic gene cluster]]s - a set of gene that contain all the necessary enzymes that lead to the production of special metabolites (formally known as [[secondary metabolite]]).<ref>{{cite journal | vauthors = Hemmerling F, Piel J | title = Strategies to access biosynthetic novelty in bacterial genomes for drug discovery | journal = Nature Reviews. Drug Discovery | volume = 21 | issue = 5 | pages = 359–378 | date = May 2022 | pmid = 35296832 | doi = 10.1038/s41573-022-00414-6 }}</ref> A benefit of using plasmids to transfer BGC is demonstrated by using a suitable host that can mass produce specialized metabolites, some of these molecules are able to control microbial population.<ref>{{cite journal | vauthors = Davies J | title = Specialized microbial metabolites: functions and origins | journal = The Journal of Antibiotics | volume = 66 | issue = 7 | pages = 361–364 | date = July 2013 | pmid = 23756686 | doi = 10.1038/ja.2013.61 }}</ref><ref name=":2" /> Plasmids can contain and express several BGCs with a few plasmids known to be exclusive for transferring BGCs.<ref name=":2">{{cite journal | vauthors = Saati-Santamaría Z | title = Global Map of Specialized Metabolites Encoded in Prokaryotic Plasmids | journal = Microbiology Spectrum | volume = 11 | issue = 4 | pages = e0152323 | date = August 2023 | pmid = 37310275 | pmc = 10434180 | doi = 10.1128/spectrum.01523-23 | editor-first = Olaya | editor-last = Rendueles }}</ref> BGC's can also be transfers to the host organism's chromosome, utilizing a plasmid vector, which allows for studies in gene knockout experiments.<ref>{{cite journal | vauthors = Okino N, Li M, Qu Q, Nakagawa T, Hayashi Y, Matsumoto M, Ishibashi Y, Ito M | title = Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and α-galactosylceramide | journal = The Journal of Biological Chemistry | volume = 295 | issue = 31 | pages = 10709–10725 | date = July 2020 | pmid = 32518167 | pmc = 7397116 | doi = 10.1074/jbc.RA120.013796 | doi-access = free }}</ref> By using plasmids for the uptake of BGCs, microorganisms can gain an advantage as production is not limited to antibiotic resistant biosynthesis genes but the production of [[toxin]]s/antitoxins.<ref>{{cite journal | vauthors = Mara P, Geller-McGrath D, Suter E, Taylor GT, Pachiadaki MG, Edgcomb VP | title = Plasmid-Borne Biosynthetic Gene Clusters within a Permanently Stratified Marine Water Column | journal = Microorganisms | volume = 12 | issue = 5 | pages = 929 | date = May 2024 | pmid = 38792759 | pmc = 11123730 | doi = 10.3390/microorganisms12050929 | doi-access = free }}</ref>