Richard Lab:Bio-Brick Formats

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Introduction
In general the Richard Lab uses the standard Bio-Brick format. However occasionally this format is incompatible with our goals (e.g. making protein fusions) or is unnecessarilly cumbersome (e.g. PCR overlap extension). To have a review on why (and how) we use these sites see the our Richard Lab:Amplified Insert Assembly page.

Biobrick Standard
This standard uses the typical biobrick assembly enzymes [EcoRI (GAATTC), XbaI (TCTAGA), SpeI (ACTAGT), and PstI (CTGCAG)] as well as NotI sites (GCGGCCGC) flanking the part. There is a slightly different prefix for protein coding parts to put the start codon in a better spot in relation to the RBS. During assembly the XbaI site of the "rear part" and the SpeI site of the "front" part eliminate eachother and form a "scar" which forever binds the two parts together.
 * Prefix
 * GAATTCgcggccgctTCTAGAg
 * The EcoRI and XbaI sites are in CAPS while the NotI site is in bold.


 * Protein Coding Prefix
 * GAATTCgcggccgctTCTAG
 * using this prefix the first base of the start codon (i.e. the "A" in "ATG") will complete the XbaI site.


 * Suffix
 * tACTAGTagcggccgCTGCAG


 * Scar
 * Tactagag for non coding parts and Tactag between coding parts and Ribosome binding sites.

Short Biobrick
Because the NotI sites in the biobricks are rarely used, a shortened biobrick system can be employed. This is especailly usefull when adding "flanking" restriction sites to sequences via overlap extention PCR. The shortened sequences are thus:
 * Prefix
 * GAATCgctTCTAGAg
 * The EcoRI and XbaI sites are in CAPS.


 * Protein Coding Prefix
 * GAATCgctTCTAG
 * using this prefix the first base of the start codon (i.e. the "A" in "ATG") will complete the XbaI site.


 * Suffix
 * tACTAGTagctCTGCAG


 * Scar
 * TACTAGAG for non coding parts and TACTAG between coding parts and Ribosome binding sites.

Protein Fusions
From time to time it becomes necessary to make protein fusions. As This is not a common occurance we use a really simple method which is not really bio-bricking. We either use HindIII (AAGCTT) or BamHI (GGATCC) as in frame restriction sites which couple with identical in frame sites in another protein. Mostly this involves attaching signal peptides to the N terminus of enzymes. In this case the modified biobrick prefix for an enzyme is: and the modified suffix for the signal peptide is:
 * GAATTCgcggccgctTCTAGAtgAAGCTT......
 * ......AAGCTTtACTAGTagcggcgCTGCAG