Registry of standard biological parts/PCR-based assembly

• Automated PCR primer design for worm genes - This is a good example of a simple CAD tool to encourage assembly. You could imagine something similar to auto-generate the primers necessary for PCR-based assembly of standard parts.
• Sauer:Stitching Genes by PCR
• patent on some of this stuff

Demo

Going to get a better feel for this by doing a few biobrick constructions using PCR-assembly

• Registry of standard biological parts/PCR-based assembly/Protocol

1. <bbpart>J04500</bbpart>+<bbpart>I13501</bbpart> = <bbpart>J04450</bbpart>
2. <bbpart>R0010</bbpart>+<bbpart>I13504</bbpart> = <bbpart>J04430</bbpart>

1. <bbpart>F2620</bbpart>+<bbpart>P1010</bbpart>+<bbpart>E0240</bbpart>
   • will try this one as a 3-way

UDG

Can use UDG (NEB USER enzyme) to replace all restriction digests and could also eliminate secondary PCRs also. You would just need to design your oligos to contain a U in the right location, assuming that there'll be a T somewhere in the vacinity of the ends of the parts you are trying to combine.

In-Fusion

• Clontech In-Fusion PCR cloning -- this could be used for the last step so that we don't need to do a digest/ligation at any point in the process.
  • Using In-fusion to combine multiple parts
• Seamless cloning and gene fusion review [1]
• Useful forum discussion
• Patents: 1, 2 (might be same as 1, title same though filing date is later), 3
• This reference describes a system similar to In-Fusion using the vaccinia virus DNA polymerase [2]

References

1. Lu Q. Seamless cloning and gene fusion. Trends Biotechnol. 2005 Apr;23(4):199-207. DOI:10.1016/j.tibtech.2005.02.008 |
2. Hamilton MD, Nuara AA, Gammon DB, Buller RM, and Evans DH. Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res. 2007;35(1):143-51. DOI:10.1093/nar/gkl1015 |
3. Horton RM, Cai ZL, Ho SN, and Pease LR. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 1990 May;8(5):528-35.
4. Yon J and Fried M. Precise gene fusion by PCR. Nucleic Acids Res. 1989 Jun 26;17(12):4895. DOI:10.1093/nar/17.12.4895 |
5. Karreman C. Fusion PCR, a one-step variant of the "megaprimer" method of mutagenesis. Biotechniques. 1998 May;24(5):736, 740, 742. DOI:10.2144/98245bm08 |
6. Wang HL, Postier BL, and Burnap RL. Optimization of fusion PCR for in vitro construction of gene knockout fragments. Biotechniques. 2002 Jul;33(1):26, 28, 30 passim. DOI:10.2144/02331bm02 |
7. Geu-Flores F, Nour-Eldin HH, Nielsen MT, and Halkier BA. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res. 2007;35(7):e55. DOI:10.1093/nar/gkm106 |
8. Donahue WF, Turczyk BM, and Jarrell KA. Rapid gene cloning using terminator primers and modular vectors. Nucleic Acids Res. 2002 Sep 15;30(18):e95. DOI:10.1093/nar/gnf094 |
9. Shevchuk NA, Bryksin AV, Nusinovich YA, Cabello FC, Sutherland M, and Ladisch S. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res. 2004 Jan 22;32(2):e19. DOI:10.1093/nar/gnh014 |
10. McDonald WF and Traktman P. Overexpression and purification of the vaccinia virus DNA polymerase. Protein Expr Purif. 1994 Aug;5(4):409-21. DOI:10.1006/prep.1994.1059 |