International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 4.4, pp. 140-142

Section 4.4.3. Cloning

K. H. Choia*

aDepartment of Biochemistry and Molecular Biology, 6.614C Basic Science, The University of Texas Medical Branch,University Blvd, Galveston, TX 77555–0647, USA
Correspondence e-mail: kychoi@utmb.edu

4.4.3. Cloning

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Development of robotics that utilize a 96-well format has changed traditional sequential cloning of individual proteins such that parallel HT cloning and protein preparation are now possible. The conventional steps in cloning are PCR amplification, restriction enzyme digestion, ligation, transformation, selection of transformers and protein expression tests. Many procedures, including amplification of target genes, cloning and screening for expression, can be achieved in parallel (96 samples at a time) using either a single liquid-handling robot or multi-channel pipettors. In 96-well parallel cloning, reaction steps for individual wells cannot be optimized, and thus care should be taken to synchronize all reactions and to minimize the number of steps to increase efficiency. For this reason, certain cloning strategies are more popular for HT cloning. Recombinant protein expression in E. coli is the most common, and will be discussed here.

4.4.3.1. Ligation-independent and recombination cloning

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Ligation-independent cloning (LIC) strategies remove the need for restriction enzyme digestion and ligation of PCR products, and are thus ideal for use in an HT cloning procedure. In LIC, PCR primers are designed to append sequences that, after treatment with T4 DNA polymerase, generate 12- to 15-base overhangs that are complementary to overhangs in the vector (Aslanidis & de Jong, 1990[link]). These longer cohesive ends make the insert–vector complex sufficiently stable to allow the transformation of hosts without ligation of the fragments.

The recombination strategy is based on the site-specific recombination reaction involved in bacteriophage λ integration and excision (Hartley et al., 2000[link]). PCR primers are designed to contain the specific sequences of recombination at 5′ and 3′ ends of the target gene, and the resulting PCR product is subcloned into a shuttle vector via site-specific recombination in the presence of integrase, integration host factor proteins and excisionase (e.g. the Gateway cloning system from Invitrogen). This clone can then either be isolated after transformation or directly used without purification for cloning into an expression vector. The major advantage of recombination cloning is that it provides a convenient way to shuttle an insert from one vector to another and thus is useful to test multiple expression conditions.

4.4.3.2. Practical application

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Since the PCR reaction must be synchronized for all 96 wells in a plate, PCR primer design is important for the success of the PCR reaction. All PCR primers should have similar melting temperatures, between 50 and 60 °C. PCR products and the prepared vector are mixed and transferred into competent cells aliquoted into a 96-well plate. The PCR products and other DNA samples during the cloning, including the restriction-enzyme digest or a plasmid preparation (if needed), can be analysed via gel electrophoresis using a commercially available 96-well agarose gel (E-gel 96 from Invitrogen) in less than 15 min. The gel is compatible with a 96-well format and can be loaded either with a liquid-handling robot or multi-channel pipettor (Fig. 4.4.3.1[link]a). The PCR step usually results in success rates as high as 98%. DNA purification kits are available in a 96-well format that use a vacuum manifold. As described above, the restriction-enzyme digest and ligation steps are not necessary in some cloning strategies.

[Figure 4.4.3.1]

Figure 4.4.3.1 | top | pdf |

Diagram of HT cloning, expression and purification. All steps can be performed in a 96-well format except the step labelled with an asterisk (*). (a) PCR amplification. PCR products are loaded onto an E-gel (1% agarose) in a 96-well format with a molecular marker (lane `M') and visualized with ethidium bromide. A close-up view of the gel outlining individual wells is also shown. (b) Transformation. Transformed cells were plated in 48-well agar plates. (c) Protein expression was tested using a western dot blot. H1 and H7 are negative and positive controls, respectively. (d) Small-scale purification using affinity resin. A 96-well filter plate in small-scale protein purification steps (left) and a 96-well pre-cast protein gel electrophoresis system (right) are shown. (e) Crystallization robot for setting up 96-well crystallization plates (right). (f) Crystal imaging and scoring system. A close-up view of an image of well B10 is shown on the right.

Following transformation, cells are then plated into two 48-grid agar growth plates mixed with appropriate antibiotics (Fig. 4.4.3.1[link]b). The 48-grid agar plate is made by inserting a cloning grill into a square Petri dish before pouring the agar, and the grill remains embedded in the media. Small glass beads can be added to each segment of the 48 grids to spread the solution. Colonies are picked by a colony picker, and are grown in 96-well deep-well blocks containing media and appropriate antibiotics. Using a liquid-handling robot and a colony picker for a single round of cloning, it is possible to achieve >80% efficiency in a 96-well format. The same robotic setup can be used for the subsequent recovery of recombinant plasmid before DNA sequencing and protein expression.

References

Aslanidis, C. & de Jong, P. J. (1990). Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074.
Hartley, J. L., Temple, G. F. & Brasch, M. A. (2000). DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795.








































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