Internal Restriction Sites
If any of the restriction enzymes you want to use are present in your gene, you need to remove them. For Biobrick 2.0 format, that would be BamHI, BglII, EcoRI, and XhoI. This section of the tutorial will explain one of many ways to achieve this.
As an example of this, let's look at a salicylate promoter basic part, Bca1111. This part confers exogenous salicylate-dependent transcription of downstream genes. Let me point out an unusual aspect of Biobrick basic parts before we dive into the construction file. A Biobrick basic part is defined as a sequence flanked by Biobrick restriction enzymes that has not been constructed based on biobrick standard assembly. In other words, anything that cannot be described as a composite part is a basic part. In the case of basic parts such as the ceaB open reading frame described in the first section, the open reading frame for the protein cannot be trivial deconstructed into a series of simpler sequences. Let's call this type of element a fundamental part. Therefore the part you designed is a "basic part" and a "fundamental part"--it can't be made any more basic. Biobrick part Bca1111, in contrast, contains several of these fundamental parts. It has an entire gene cassette of a promoter, ribosome binding site, the nahR open reading frame, and terminator. Together these parts produce the transcription factor protein, NahR. Additionally, Bca1111 contains the Psal promoter, which is activated by NahR. We could Biobrick all these fundamental parts, and then assemble something with the same utility as Bca1111 as a composite part. This procedure is the essence of refactoring--splitting a naturally-occuring sequence into its fundamental basic parts and reassembling them into a composite part that maintains the activity of the original. In some instances, this might be useful as subtle properties of the original cassette could be altered or improved by refactoring. In other instances, it just creates more work for you.
Alright, let's look at the construction file:
PCR ca1110F/ca1111R on pBACr899 (814 bp, gp = A) PCR ca1111F/ca899R on pBACr899 (497 bp, gp = B) --------------------------------------------------- PCR ca1110F/ca899R on A+B (1287 bp, EcoRI/BamHI) Digest pBca1100 (EcoRI/BamHI, 2927+28, L Product is pBca1100-Bca1111