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| Optimization and Application of SLiCE in vitro Assembly Method |
| FENG Qi1, WANG Ying1,2 |
1. Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, China;
2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China |
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Abstract Seamless ligation cloning extract (SLiCE) is a seamless ligation method which utilizes cell extracts to assemble DNA fragments in vitro through recombination reaction. Here, this method was further optimized by identifying the sources of cell extract, enhancing the expression of λ recombination system and adjusting the ratio of fragments used in assembly system. The cell extract from Saccharomyces cerevisiae and Bacillus subtilis were proved to be not suitable for assembly in vitro. The plasmid with λ recombination system was introduced into Escherichia coli DH10B, and the cell extract from this strain was proved to be effective for assembly in vitro. A color-based screen method was constructed to identify positive clones and measure the accuracy in the 3-fragments assembly assay. Finally, a melanin expression cassette, which can be expressed in S. cerevisiae, was assembled via the SLiCE method optimized.
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Received: 05 June 2015
Published: 25 October 2015
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| [1] Andrianantoandro E, Basu S, Karig D K, et al. Synthetic biology: new engineering rules for an emerging discipline. Mol Syst Biol, 2006, 2(1):1-14.
[2] Ellis T, Adie T, Baldwin G S, DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol (Camb), 2011, 3(2): 109-118.
[3] Smolke C D. Building outside of the box: IGEM and the BioBricks foundation. Nat Biotechnol, 2009, 27(12): 1099-1102.
[4] Anderson J C, Dueber J E, Leguia M, et al. BglBricks: a flexible standard for biological part assembly. J Biol Eng, 2010, 4(1): 1-12.
[5] Horton R M, Hunt H D, Ho S N, et al. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene, 1989, 77(1): 61-68.
[6] Zhu B I, Cai G, Hall E O, et al. In-FusionTM assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques, 2007, 43(3): 354-359.
[7] Gibson D G, Smith H O, Hutchison C A, et al. Chemical synthesis of the mouse mitochondrial genome. Nat Methods, 2010, 11(7): 901-905.
[8] Zhang Y, Werling U, Edelmann W. SLiCE: A novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res, 2012, 40(8): e55.
[9] Fisher A B, Canfield Z B, Hayward L C, et al. Ex Vivo DNA assembly. Front Bioeng Biotechnol, 2013, 1(12):1-7.
[10] Itaya M, Fujita K, Kuroki A, et al. Bottom-up genome assembly using the Bacillus subtilis genome vector. Nat Methods, 2007, 5 (1): 41-43.
[11] Murphy K C. Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J Bacteriol, 1998, 180(8): 2063-2071.
[12] Claus H, Decker H. Bacterial tyrosinases. Syst Appl Microbiol, 2006, 29(1): 3-14.
[13] Lagunas-Munoz V H, Cabrera-Valladares N, Bolivar F, et al. Optimum melanin production using recombinant Escherichia coli. J Appl Microbiol, 2006, 101(5): 1002-1008. |
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