Template:SBB09 Cterm2DisplayNotes



This part encodes a C-terminal display protein
Your displayer part should be of the {a~part>} style (no stop). The tilda business is our preferred way of encoding the rbs/cds junction. Basically, the BglBrick scar (GGATCT) will end up superimposed over the Shine-Delgarno sequence. So, the tilda refers to the inclusion of 4 addition bases before the start codon, and the "a" refers to the fact that you'll make your part have an ATG start codon.

You'll need to include part of the native 5'UTR and prepro region of your protein in the part. To do this, you first of all need the complete sequence of your protein and it's 5' UTR (the region upstream of the start codon). To illustrate this, here's an example:

You can download the raw sequence of the fimH region annotated [[Media:JCA_fimH_region.seq | here]].

Locate the start codon and make sure it's the real start. Sometimes starts are GTG or TTG! If it is TTG or GTG, change it to ATG. Now, include the next four 5' bases and then put in your BglII site.

So, the start for this fimH part is: atgaaacgagttattaccc...

The next 4 upstream bases are TGTA, so my new sequence is: AGATCTtgtaatgaaacgagttattaccc

and the forward oligo would be something like: ccaaaGAATTCatgAGATCTtgtaatgaaacgagttattaccctg

You should design your construction file to insert your part into plasmid pBca9495CA-Bca1144#5 using EcoRI and BamHI. The map of this plasmid is [[Media:JCA_pBca9495CA-Bca1144.seq | here]].