In-Fusion BioBrick assembly and re-engineering.
Genetic circuits can be assembled from standardized biological parts called BioBricks. Examples of BioBricks include promoters, ribosome-binding sites, coding sequences and transcriptional terminators. Standard BioBrick assembly normally involves restriction enzyme digestion and ligation of two BioBricks at a time. The method described here is an alternative assembly strategy that allows for two or more PCR-amplified BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit. This method allows for a large number of parallel assemblies to be performed and is a flexible way to mix and match BioBricks. In-Fusion assembly can be semi-standardized by the use of simple primer design rules that minimize the time involved in planning assembly reactions. We describe the success rate and mutation rate of In-Fusion assembled genetic circuits using various homology and primer lengths. We also demonstrate the success and flexibility of this method with six specific examples of BioBrick assembly and re-engineering. These examples include assembly of two basic parts, part swapping, a deletion, an insertion, and three-way In-Fusion assemblies.
Sleight
Sean C
SC
Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. sleight@u.washington.edu
Bartley
Bryan A
BA
Lieviant
Jane A
JA
Sauro
Herbert M
HM
D004247:DNA / Q000737:chemistry; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013728:Terminator Regions, Genetic
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D004247:DNA
J Am Chem Soc. 2009 May 13;131(18):6508-15
19378995
Biotechnol Bioeng. 2005 Dec 5;92(5):553-8
16247774
Nucleic Acids Res. 2010 May;38(8):2624-36
20385581
Biotechniques. 2007 Sep;43(3):354-9
17907578
Proc Natl Acad Sci U S A. 1990 Oct;87(19):7668-72
2217199
J Biol Eng. 2008 Apr 14;2:5
18410688
Nucleic Acids Res. 2007;35(7):e55
17389646
Nat Methods. 2007 Mar;4(3):251-6
17293868
PLoS One. 2009;4(5):e5553
19436741
Protein Expr Purif. 2006 Jan;45(1):66-71
16289702
Nat Methods. 2009 May;6(5):343-5
19363495
J Biol Eng. 2009 Mar 20;3:4
19298678
Biotechniques. 1990 May;8(5):528-35
2357375
Nucleic Acids Res. 2009 Nov;37(20):6984-90
19745056
Protein Eng Des Sel. 2010 Jan;23(1):1-8
19897542
Nat Biotechnol. 2008 Jul;26(7):787-93
18612302
J Biol Eng. 2010 Jan 20;4(1):1
20205762