1887
No metrics data to plot.
The attempt to load metrics for this article has failed.
The attempt to plot a graph for these metrics has failed.

DNA Assembly Tools and Strategies for the Generation of Plasmids

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
  • HTML
    100.73 Kb
  • XML
    96.78 Kb
  • PDF
    345.32 Kb
  • Authors: Chang-Ho Baek1, Michael Liss2, Kevin Clancy3, Jonathan Chesnut4, Federico Katzen5
  • Editors: Marcelo E. Tolmasky6, Juan Carlos Alonso7
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Life Technologies, Carlsbad, CA 92008; 2: Life Technologies, Carlsbad, CA 92008; 3: Life Technologies, Carlsbad, CA 92008; 4: Life Technologies, Carlsbad, CA 92008; 5: Life Technologies, Carlsbad, CA 92008; 6: California State University, Fullerton, CA; 7: Centro Nacional de Biotecnología, Cantoblanco, Madrid, Spain
  • Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0014-2013
  • Received 02 December 2013 Accepted 18 December 2013 Published 24 October 2014
  • Federico Katzen, Federico.Katzen@lifetech.com.
image of DNA Assembly Tools and Strategies for the Generation of Plasmids
    Preview this microbiology spectrum article:
    Zoom in
    Zoomout

    DNA Assembly Tools and Strategies for the Generation of Plasmids, Page 1 of 2

    | /docserver/preview/fulltext/microbiolspec/2/5/PLAS-0014-2013-1.gif /docserver/preview/fulltext/microbiolspec/2/5/PLAS-0014-2013-2.gif
  • Abstract:

    Since the discovery of restriction enzymes and the generation of the first recombinant DNA molecule over 40 years ago, molecular biology has evolved into a multidisciplinary field that has democratized the conversion of a digitized DNA sequence stored in a computer into its biological counterpart, usually as a plasmid, stored in a living cell. In this article, we summarize the most relevant tools that allow the swift assembly of DNA sequences into useful plasmids for biotechnological purposes. We cover the main components and stages in a typical DNA assembly workflow, namely design, gene synthesis, and and sequence assembly methodologies.

  • Citation: Baek C, Liss M, Clancy K, Chesnut J, Katzen F. 2014. DNA Assembly Tools and Strategies for the Generation of Plasmids. Microbiol Spectrum 2(5):PLAS-0014-2013. doi:10.1128/microbiolspec.PLAS-0014-2013.

Key Concept Ranking

Genetic Elements
0.5182467
DNA Synthesis
0.48029897
Circular Double-Stranded DNA
0.40913272
0.5182467

References

1. Jackson DA, Symons RH, Berg P. 1972. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc Natl Acad Sci USA 69:2904–2909. [PubMed][CrossRef]
2. Lu Q. 2005. Seamless cloning and gene fusion. Trends Biotechnol 23:199–207. [PubMed][CrossRef]
3. Ellis T, Adie T, Baldwin GS. 2011. DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol (Camb) 3:109–118. [PubMed][CrossRef]
4. Durani V, Sullivan BJ, Magliery TJ. 2012. Simplifying protein expression with ligation-free, traceless and tag-switching plasmids. Protein Expr Purif 85:9–17. [PubMed][CrossRef]
5. Tolmachov O. 2009. Designing plasmid vectors. Methods Mol Biol 542:117–129. [PubMed][CrossRef]
6. Wang X, Sa N, Tian PF, Tan TW. 2011. Classifying DNA assembly protocols for devising cellular architectures. Biotechnol Adv 29:156–163. [PubMed][CrossRef]
7. Green MR, Sambrook J. 2012. Molecular Cloning: A Laboratory Manual, 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
8. Preston A. 2003. Choosing a cloning vector. Methods Mol Biol 235:19–26. [PubMed]
9. del Solar G, Giraldo R, Ruiz-Echevarria MJ, Espinosa M, Diaz-Orejas R. 1998. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev 62:434–464. [PubMed]
10. Scott JR. 1984. Regulation of plasmid replication. Microbiol Rev 48:1–23. [PubMed]
11. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S. 1977. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113. [PubMed][CrossRef]
12. Vieira J, Messing J. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268. [PubMed][CrossRef]
13. Hashimoto-Gotoh T, Timmis KN. 1981. Incompatibility properties of Col E1 and pMB1 derivative plasmids: random replication of multicopy replicons. Cell 23:229–238. [PubMed][CrossRef]
14. Kues U, Stahl U. 1989. Replication of plasmids in gram-negative bacteria. Microbiol Rev 53:491–516. [PubMed]
15. Ramsay M. 1994. Yeast artificial chromosome cloning. Mol Biotechnol 1:181–201. [PubMed][CrossRef]
16. Den Dunnen JT, Grootscholten PM, Dauwerse JG, Walker AP, Monaco AP, Butler R, Anand R, Coffey AJ, Bentley DR, Steensma HY, et al. 1992. Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. Hum Mol Genet 1:19–28. [PubMed][CrossRef]
17. Loenen WA, Raleigh EA. 2014. The other face of restriction: modification-dependent enzymes. Nucleic Acids Res 42:56–69. [PubMed][CrossRef]
18. Zhou MY, Clark SE, Gomez-Sanchez CE. 1995. Universal cloning method by TA strategy. Biotechniques 19:34–35. [PubMed]
19. Shuman S. 1994. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem 269:32678–32684. [PubMed]
20. Hartley JL, Temple GF, Brasch MA. 2000. DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795. [PubMed][CrossRef]
21. Cheo DL, Titus SA, Byrd DR, Hartley JL, Temple GF, Brasch MA. 2004. Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. Genome Res 14:2111–2120. [PubMed][CrossRef]
22. Buchholz F, Bishop M. 2001. LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme. Biotechniques 31:906–908, 910, 912, 914, 916, 918. [PubMed]
23. Zhou MY, Gomez-Sanchez CE. 2000. Universal TA cloning. Curr Issues Mol Biol 2:1–7. [PubMed]
24. Katzen F. 2007. Gateway® recombinational cloning: a biological operating system. Expert Opin Drug Discov 2:571–589. [PubMed][CrossRef]
25. Knight TF. 2003. Idempotent Vector Design for Standard Assembly of BioBricks. MIT Synthetic Biology Working Group Technical Reports. http://hdl.handle.net/1721.1/21168.
26. Norville JE, Derda R, Gupta S, Drinkwater KA, Belcher AM, Leschziner AE, Knight TF, Jr. 2010. Introduction of customized inserts for streamlined assembly and optimization of BioBrick synthetic genetic circuits. J Biol Eng 4:17. doi:10.1186/1754-1611-4-17. [PubMed][CrossRef]
27. Engler C, Kandzia R, Marillonnet S. 2008. A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647. doi:10.1371/journal.pone.0003647. [PubMed][CrossRef]
28. Engler C, Gruetzner R, Kandzia R, Marillonnet S. 2009. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4:e5553. doi:10.1371/journal.pone.0005553. [PubMed][CrossRef]
29. Engler C, Marillonnet S. 2011. Generation of families of construct variants using golden gate shuffling. Methods Mol Biol 729:167–181. [PubMed][CrossRef]
30. Weber E, Gruetzner R, Werner S, Engler C, Marillonnet S. 2011. Assembly of designer TAL effectors by Golden Gate cloning. PLoS One 6:e19722. doi:10.1371/journal.pone.0019722. [PubMed][CrossRef]
31. Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juarez P, Fernandez-del-Carmen A, Granell A, Orzaez D. 2011. GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules. PLoS One 6:e21622. doi:10.1371/journal.pone.0021622. [PubMed][CrossRef]
32. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. 2011. A modular cloning system for standardized assembly of multigene constructs. PLoS One 6:e16765. doi:10.1371/journal.pone.0016765. [PubMed][CrossRef]
33. Wang RY, Shi ZY, Guo YY, Chen JC, Chen GQ. 2013. DNA fragments assembly based on nicking enzyme system. PLoS One 8:e57943. doi:10.1371/journal.pone.0057943. [PubMed][CrossRef]
34. Chen WH, Qin ZJ, Wang J, Zhao GP. 2013. The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly. Nucleic Acids Res 41:e93. doi:10.1093/nar/gkt122. [PubMed][CrossRef]
35. Aslanidis C, de Jong PJ. 1990. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074. [PubMed][CrossRef]
36. Li MZ, Elledge SJ. 2007. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256. [PubMed][CrossRef]
37. Zhu B, Cai G, Hall EO, Freeman GJ. 2007. In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43:354–359. [PubMed][CrossRef]
38. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. [PubMed][CrossRef]
39. Tsvetanova B, Peng L, Liang X, Li K, Yang JP, Ho T, Shirley J, Xu L, Potter J, Kudlicki W, Peterson T, Katzen F. 2011. Genetic assembly tools for synthetic biology. Methods Enzymol 498:327–348. [PubMed][CrossRef]
40. Bitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R, Vaiskunaite R. 2007. USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res 35:1992–2002. [PubMed][CrossRef]
41. Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U. 2010. Phosphorothioate-based ligase-independent gene cloning (PLICing): an enzyme-free and sequence-independent cloning method. Anal Biochem 406:141–146. [PubMed][CrossRef]
42. Marienhagen J, Dennig A, Schwaneberg U. 2012. Phosphorothioate-based DNA recombination: an enzyme-free method for the combinatorial assembly of multiple DNA fragments. Biotechniques 0:1–6.
43. Coljee VW, Murray HL, Donahue WF, Jarrell KA. 2000. Seamless gene engineering using RNA- and DNA-overhang cloning. Nat Biotechnol 18:789–791. [PubMed][CrossRef]
44. Miyazaki K, Takenouchi M. 2002. Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. Biotechniques 33:1033–1034, 1036–1038. [PubMed]
45. Quan J, Tian J. 2011. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc 6:242–251. [PubMed][CrossRef]
46. Zhang Y, Buchholz F, Muyrers JP, Stewart AF. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128. [PubMed][CrossRef]
47. Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Muller R, Stewart AF, Zhang Y. 2012. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol 30:440–446. [PubMed][CrossRef]
48. Itaya M, Fujita K, Kuroki A, Tsuge K. 2008. Bottom-up genome assembly using the Bacillus subtilis genome vector. Nat Methods 5:41–43. [PubMed][CrossRef]
49. Kruger NJ, Stingl K. 2011. Two steps away from novelty--principles of bacterial DNA uptake. Mol Microbiol 80:860–867. [PubMed][CrossRef]
50. Gibson DG, Benders GA, Axelrod KC, Zaveri J, Algire MA, Moodie M, Montague MG, Venter JC, Smith HO, Hutchison CA, 3rd. 2008. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci USA 105:20404–20409. [PubMed][CrossRef]
51. Tagwerker C, Dupont CL, Karas BJ, Ma L, Chuang RY, Benders GA, Ramon A, Novotny M, Montague MG, Venepally P, Brami D, Schwartz A, Andrews-Pfannkoch C, Gibson DG, Glass JI, Smith HO, Venter JC, Hutchison CA, 3rd. 2012. Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast. Nucleic Acids Res 40:10375–10383. [PubMed][CrossRef]
52. Larionov V, Kouprina N, Eldarov M, Perkins E, Porter G, Resnick MA. 1994. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks. Yeast 10:93–104. [PubMed][CrossRef]
53. Raymond CK, Sims EH, Olson MV. 2002. Linker-mediated recombinational subcloning of large DNA fragments using yeast. Genome Res 12:190–197. [PubMed][CrossRef]
54. Gibson DG. 2009. Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37:6984–6990. [PubMed][CrossRef]
55. Wingler LM, Cornish VW. 2011. Reiterative Recombination for the in vivo assembly of libraries of multigene pathways. Proc Natl Acad Sci USA 108:15135–15140. [PubMed][CrossRef]
56. Braman J. 2002. In Vitro Mutagenesis Protocols. Humana Press, New York City, NY. [CrossRef]
57. Hutchison CA, 3rd, Phillips S, Edgell MH, Gillam S, Jahnke P, Smith M. 1978. Mutagenesis at a specific position in a DNA sequence. J Biol Chem 253:6551–6560. [PubMed]
58. Zoller MJ, Smith M. 1987. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol 154:329–350. [PubMed][CrossRef]
59. Hogrefe HH, Cline J, Youngblood GL, Allen RM. 2002. Creating randomized amino acid libraries with the QuikChange Multi Site-Directed Mutagenesis Kit. Biotechniques 33:1158–1160, 1162, 1164–1155. [PubMed]
60. Bauer JC, Wright DA, Braman JC, Geha RS. 1998. Circular site-directed mutagenesis. US patent 5789166, August.
61. Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL. 1995. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53. [PubMed][CrossRef]
62. Kunkel TA. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492. [PubMed][CrossRef]
63. Hemsley A, Arnheim N, Toney MD, Cortopassi G, Galas DJ. 1989. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res 17:6545–6551. [PubMed][CrossRef]
64. Chen Z, Ruffner DE. 1998. Amplification of closed circular DNA in vitro. Nucleic Acids Res 26:1126–1127. [CrossRef]
65. Liang X, Peng L, Li K, Peterson T, Katzen F. 2012. A method for multi-site-directed mutagenesis based on homologous recombination. Anal Biochem 427:99–101. [PubMed][CrossRef]
66. Agarwal KL, Buchi H, Caruthers MH, Gupta N, Khorana HG, Kleppe K, Kumar A, Ohtsuka E, Rajbhandary UL, Van de Sande JH, Sgaramella V, Weber H, Yamada T. 1970. Total synthesis of the gene for an alanine transfer ribonucleic acid from yeast. Nature 227:27–34. [PubMed][CrossRef]
67. Mandecki W, Hayden MA, Shallcross MA, Stotland E. 1990. A totally synthetic plasmid for general cloning, gene expression and mutagenesis in Escherichia coli. Gene 94:103–107. [PubMed][CrossRef]
68. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA, 3rd, Smith HO, Venter JC. 2010. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56. [PubMed][CrossRef]
69. Raab D, Graf M, Notka F, Schodl T, Wagner R. 2010. The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst Synth Biol 4:215–225. [PubMed][CrossRef]
70. Graf M, Schoedl T, Wagner R. 2009. Rationales of gene design and de novo gene construction, p 411–438. In Fu P, Panke S (ed), Systems Biology and Synthetic Biology, vol 1. John Wiley & Sons Inc, Hoboken, NJ. [CrossRef]
71. Greger B, Kemper B. 1998. An apyrimidinic site kinks DNA and triggers incision by endonuclease VII of phage T4. Nucleic Acids Res 26:4432–4438. [PubMed][CrossRef]
72. Smith J, Modrich P. 1997. Removal of polymerase-produced mutant sequences from PCR products. Proc Natl Acad Sci USA 94:6847–6850. [PubMed][CrossRef]
73. Young L, Dong Q. 2004. Two-step total gene synthesis method. Nucleic Acids Res 32:e59. doi:10.1093/nar/gnh058. [PubMed][CrossRef]
74. Padgett KA, Sorge JA. 1996. Creating seamless junctions independent of restriction sites in PCR cloning. Gene 168:31–35. [PubMed][CrossRef]
75. Mullinax RL, Gross EA, Hay BN, Amberg JR, Kubitz MM, Sorge JA. 1992. Expression of a heterodimeric Fab antibody protein in one cloning step. Biotechniques 12:864–869. [PubMed]
76. Dietmaier W, Fabry S, Schmitt R. 1993. DISEC-TRISEC: di- and trinucleotide-sticky-end cloning of PCR-amplified DNA. Nucleic Acids Res 21:3603–3604. [PubMed][CrossRef]
77. Clancy K, Voigt CA. 2010. Programming cells: towards an automated ‘Genetic Compiler.’ Curr Opin Biotechnol 21:572–581. [PubMed][CrossRef]
78. Chen YJ, Clancy K, Voigt CA. 2012. Modeling genetic parts for synthetic biology, p 197–231. In Wall ME (ed), Quantitative Biology: From Molecular to Cellular Systems, vol 1. CRC Press, Boca Raton, FL.
79. D. C, Bergmann FT, Sauro HM, Densmore D. 2011. Computer-aided design for synthetic biology, p 203–224. In Koeppl H, Densmore D, Setti G, di Bernardo M (ed), Design and Analysis of Biomolecular Circuits. Engineering Approaches to Systems and Synthetic Biology, vol 1. Springer, New York, NY.
80. Chang AC, Cohen SN. 1978. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156. [PubMed]
81. Cohen SN, Chang AC, Boyer HW, Helling RB. 1973. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA 70:3240–3244. [PubMed][CrossRef]
82. Thomas CM, Smith CA. 1987. Incompatibility group P plasmids: genetics, evolution, and use in genetic manipulation. Annu Rev Microbiol 41:77–101. [PubMed][CrossRef]
83. Keen NT, Tamaki S, Kobayashi D, Trollinger D. 1988. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191–197. [PubMed][CrossRef]
84. Rawlings DE, Tietze E. 2001. Comparative biology of IncQ and IncQ-like plasmids. Microbiol Mol Biol Rev 65:481–496. [PubMed][CrossRef]
85. Kontomichalou P, Mitani M, Clowes RC. 1970. Circular R-factor molecules controlling penicillinase synthesis, replicating in Escherichia coli under either relaxed or stringent control. J Bacteriol 104:34–44. [PubMed]
86. Murray JA. 1987. Bending the rules: the 2-mu plasmid of yeast. Mol Microbiol 1:1–4. [PubMed][CrossRef]
87. Sikorski RS, Hieter P. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27. [PubMed]
88. Bitoun R, Zamir A. 1986. Spontaneous amplification of yeast CEN ARS plasmids. Mol Gen Genet 204:98–102. [PubMed][CrossRef]
89. Burke DT, Carle GF, Olson MV. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806–812. [PubMed][CrossRef]
microbiolspec.PLAS-0014-2013.citations
cm/2/5
content/journal/microbiolspec/10.1128/microbiolspec.PLAS-0014-2013
Loading

Citations loading...

Loading

Article metrics loading...

/content/journal/microbiolspec/10.1128/microbiolspec.PLAS-0014-2013
2014-10-24
2017-09-21

Abstract:

Since the discovery of restriction enzymes and the generation of the first recombinant DNA molecule over 40 years ago, molecular biology has evolved into a multidisciplinary field that has democratized the conversion of a digitized DNA sequence stored in a computer into its biological counterpart, usually as a plasmid, stored in a living cell. In this article, we summarize the most relevant tools that allow the swift assembly of DNA sequences into useful plasmids for biotechnological purposes. We cover the main components and stages in a typical DNA assembly workflow, namely design, gene synthesis, and and sequence assembly methodologies.

Highlighted Text: Show | Hide
Loading full text...

Full text loading...

/deliver/fulltext/microbiolspec/2/5/PLAS-0014-2013.html?itemId=/content/journal/microbiolspec/10.1128/microbiolspec.PLAS-0014-2013&mimeType=html&fmt=ahah

Figures

Image of FIGURE 1

Click to view

FIGURE 1

Schematic representation of different DNA assembly methodologies. Golden Gate Cloning. Fragments to be assembled (red and green) have strategically placed terminal type IIS endonuclease recognition sites (in this case saI sites shown in lowercase and underlined). Black arrowheads point toward the saI cleavage sites. Simultaneous incubation with BsaI and DNA ligase results in covalently linked fragments. Chew-back and repair-based assembly. Adjacent DNA fragments (red and green) sharing terminal sequence overlaps are incubated with DNA exonuclease, thereby exposing complementary DNA strands. The strands are annealed and the gaps can be sealed either by DNA polymerase and DNA ligase, or by the cell upon transformation. USER assembly. Adjacent fragments (red and green), amplified with compatible uracil-containing primers, are incubated with the USER enzyme mix, which removes the uracils. The small terminal complementary DNA strands (in black) anneal to each other, outcompeting the small terminal loose strand. Gaps are repaired and sealed by the cell upon transformation. doi:10.1128/microbiolspec.PLAS-0014-2013.f1

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0014-2013
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2

Click to view

FIGURE 2

Schematic overview of gene synthesis workflow. designed sequence data are converted into a set of oligonucleotides by automated organic chemistry. These are stepwise assembled, elongated, and amplified into a full-length fragment (see box), which is then ligated into a cloning vector. After transformation, colonies are screened for error-free insert sequences and a correct colony is cultivated for plasmid isolation. After a final sequence verification of the plasmid preparation, the construct is ready to be used or to be further assembled into larger constructs. doi:10.1128/microbiolspec.PLAS-0014-2013.f2

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0014-2013
Permissions and Reprints Request Permissions
Download as Powerpoint

Tables

Generic image for table

Click to view

TABLE 1

Replicons most commonly used in plasmids

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0014-2013
Generic image for table

Click to view

TABLE 2

Examples of commercial site-directed mutagenesis kits

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0014-2013
Generic image for table

Click to view

TABLE 3

Most commonly used bioinformatics software

Source: microbiolspec October 2014 vol. 2 no. 5 doi:10.1128/microbiolspec.PLAS-0014-2013

Supplemental Material

No supplementary material available for this content.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error