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Revision as of 17:58, 11 July 2014
Biobrick Primers for Site-Directed Insertion Mutagenesis
Ribosomal Binding Site Insertion
Purpose
The RBS primer is a standard primer that can be used for site directed mutagenesis in all linearized backbones available from the iGEM database.
Background
Most of the BioBrick parts requires a Ribosomal Binding Site to express the protein. This has proven problematic as the RBS is very small and hard to insert using 3A Assembly. We believe the creation of an oligo primer with this RBS may be easier to place the RBS in front of the protein that is needed to be expressed. An easy oligo primer that could be used in PCR extension and then placed into a vector backbone would save time and frustration as to whether the part had been inserted.
Design
The format of this primer needs the BioBrick formatted prefix with an additional strong Ribosomal binding site within it. The prefix of the standard BioBrick was analyzed and the primer was found to be able to be inserted after the XbaI site and still have the scar intact between the part and the RBS site. The RBS sites chosen were selected from the Anderson RBS library degenerate sequence and a library was created with that degenerate sequence via the Salis Lab at Penn State University, optimized for E. coli 16s.
Promoter Sequence Insertion
Purpose
The Promoter primer is a standard primer that can be used for site directed mutagenesis in all linearized backbones available from the iGEM database and the Anderson RBS sites from the Anderson RBS library.
Background
Most of the BioBrick parts requires a Promoter to express the protein. This has proven problematic as the promoter is very small and hard to insert using 3A Assembly. We believe the creation of an oligo primer with this promoter may be easier to place the promoter in front of the RBS site that is needed to be expressed. An easy oligo primer that could be used in PCR extension and then placed into a vector backbone would save time and frustration as to whether the part had been inserted.
Design
The format of this primer needs the BioBrick formatted prefix, the promoter and part of the RBS site. The prefix of the standard BioBrick was analyzed and the primer was found to be able to be inserted after the XbaI site and be able to insert the promoter just before the RBS site. The promoters were selected from the Anderson Constitutive Promoter Library in the iGEM parts registry.
