Chapter 30 : DNA Assembly Tools and Strategies for the Generation of Plasmids

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Recombinant plasmids are possibly the biological reagents most frequently used in molecular biology. Myriad plasmid assembly strategies have been devised since the first recombinant DNA molecule was generated over 40 years ago ( ). Cloning protocols are now so profuse that it is not always trivial to choose one that is the most suitable for a particular purpose. The aspects to consider at the time of choosing an assembly strategy include, among others, the nature of the sequences, fragment size and number, template availability, plasmid capacity and stability, and selection against a background of other unwanted assemblies.

Citation: Baek C, Liss M, Clancy K, Chesnut J, Katzen F. 2015. DNA Assembly Tools and Strategies for the Generation of Plasmids, p 601-613. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0014-2013
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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.

Citation: Baek C, Liss M, Clancy K, Chesnut J, Katzen F. 2015. DNA Assembly Tools and Strategies for the Generation of Plasmids, p 601-613. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0014-2013
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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.

Citation: Baek C, Liss M, Clancy K, Chesnut J, Katzen F. 2015. DNA Assembly Tools and Strategies for the Generation of Plasmids, p 601-613. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0014-2013
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Table 1

Replicons most commonly used in plasmids

Citation: Baek C, Liss M, Clancy K, Chesnut J, Katzen F. 2015. DNA Assembly Tools and Strategies for the Generation of Plasmids, p 601-613. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0014-2013
Generic image for table
Table 2

Examples of commercial site-directed mutagenesis kits

Citation: Baek C, Liss M, Clancy K, Chesnut J, Katzen F. 2015. DNA Assembly Tools and Strategies for the Generation of Plasmids, p 601-613. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0014-2013
Generic image for table
Table 3

Most commonly used bioinformatics software

Citation: Baek C, Liss M, Clancy K, Chesnut J, Katzen F. 2015. DNA Assembly Tools and Strategies for the Generation of Plasmids, p 601-613. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0014-2013

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