Difference between revisions of "Part:BBa K215200"

Latest revision as of 18:53, 1 October 2009

Generic Surface Display Construct: IPTG Inducible Lpp + 1tmr OmpA + NheI Site

This is a generic surface display construct that displays your protein of interest using the OmpA surface display system as described in [cite]. The difference between K215200 and K215201 is that K215200 attaches one transmembrane helix to your protein to anchor it into the outer membrane, and K215201 attaches an anchor that is composed of five transmembrane helices.

To insert a gene from a BioBrick (that has the standard suffix following the gene), you will need to design a primer that removes the start codon and adds a XbaI site that keeps the rest of the gene in frame with OmpA. If you are creating the gene from scratch, BioBrick it and then follow this guide so that there is the standard suffix following the gene.

For example, assume our gene (E0040) starts with: 5'-atgcgtaaaggagaagaacttt...-3'

The design process would be:

1. Start with the XbaI site: 5'-TCTAGA-3'
2. Add ~20bp of the gene immediately after the XbaI site, starting _after_ the start codon (atg), and try to end on a G or C: 5'-TCTAGAcgtaaaggagaagaacttt-3' for E0040 from above. These ~20bp will determine the melting temperature for the primer.
3. Add 6-8 random nucleotides at the start. Try to balance out the primer to ~%50 g&c to a&t: 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
4. Tweak the number of nucleotides until the melting point roughly matches that of Vr.

Then to add the gene to the construct, again assuming the gene is a BioBrick with standard suffix:

1. PCR with the designed forward primer and Vr
2. Then run the PCR product in a digest with XbaI and PstI, and also digest this part (the display construct) with NheI and PstI. The XbaI site has a sticky end that binds with NheI.
3. Standard ligation and transformation.
4. Your gene will now be expressed as a fusion protein that will be displayed, using the OmpA surface display system, on the surface of the cell.

Sequence and Features