Template:SBB09 18094

Hank Shih

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

Intimin (native)

 Source:  E. coli strain O157:H7

For background, you should read PMID: 11717287. You will be making a part derived from the eaeA gene which encodes the Intimin protein. You can download an annotated sequence here. You will be making an "a~" style part out of the green and pink regions of this file.

Note: You will be sharing the reverse oligo with whoever gets the SBB09_19607 project. You should find this person and design that oligo together.

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.

Fast-Degrading GFP

 Source: pBca1256-Bjh1857     {<GFP-LVA>}

This part has already been constructed as a BglBricks basic part in plasmid pBca1256. You will be transferring the part using the Gateway reaction into the assembly vector. To do this, you will react the source plasmid with plasmid pBca1254AK. As background, you should read PMID: 11059491.

This part encodes a passenger protein

After performing the Gateway reaction, your product plasmid will be pBca9495AK-partname. To see what the part looks like in the product vector, look at this map of the RFP-expressing part Bca1144 in pBca9495AK.