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Patrick Harrigan

Welcome to your project page!

For each part listed, you should:

• Design oligos to make your part
• Write up proper construction files and put it on the Construction Files page
• Put your oligos on the Oligo Log page
• Put your part sequence on the part document
• Name your parts according to the list here

TraA Display

First of all, read PMID: 9356245. You are going to make a basic part out of

 Source:  pOX38

You will be making a C-terminal display system out of the TraA protein from F plasmid. pOX38 is just a particular variant of the F plasmid. You should read PMID: 9356245 for background. Note that any devices containing the part will require an F+ strain for proper expression.

The sequence of TraA is available at NP_061453 in pubmed.

You can use the online tool at http://phobius.cbr.su.se/ to locate the prepro sequence.

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 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 here.

Leucine Zipper peptide KILR

 Source: Synthetic

You should read PMID: 12459719. You will make a BglBrick part encoding:

 VKELEDKNEELLSkiYHlrNEVARLKKLVGERGGC

No start, no stop, in frame.

This part encodes a passenger protein

Your passenger part should be of the {<part>} style (no start, no stop). There should be NO prepro sequence in your part. If your part is naturally secreted, you should encode only the active peptide. You should design your construction file to insert your part into plasmid pBca9495AK-Bca1144#5 using EcoRI and BamHI. The map of this plasmid is here.