Template:SBB09 19395

Your Name

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

Invasin (native, short)

 Source: plasmid pAC-TetInvdBg

You should read PMID: 11137298 for some background. You will be constructing parts derived from the inv gene of Yersinia pseudotuburculosis. The source for your parts is a plasmid, and the sequence is here. You are going to construct two parts derived from Invasin. Your forward oligo will be reused in the two construction files. For the first part, the short version, you should make an "a~" part that begins from the native start (start of red region in the plasmid map file) and extends up to the lime green region (but doesn't include any of the lime green sequence).

Due to the large size of this part, you will not sequence every base of your plasmid. You should only sequence with ca998 and G00101 (forward and reverse) and confirm the sequence of the 5' and 3' 500 bp or so.

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.

Invasin (native, long)

 Source: plasmid pAC-TetInv

Your second part is much like the first, but you will include the lime green region in the part.

Due to the large size of this part, you will not sequence every base of your plasmid. You should only sequence with ca998 and G00101 (forward and reverse) and confirm the sequence of the 5' and 3' 500 bp or so.

Note: The reverse oligo for this part is the same oligo being used for the invasin part in project SBB09_41163. You should coordinate with the person who got that project in designing a single oligo for both parts.

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.