Log050908

From 2006.igem.org

Not much time for logs these days. Currently we know what zincfinger sequencies we are ordering for the NOR module. The input-module can be cloned with the contents of the box.

However, in the teammeeting the input-module group addressed their concern about putting the zincfinger binding sites between Start and RBS. The concern is, that the Polymerase might be binding anyway and open the helical structure and thus the break the ZF-binding to the helical structure (it only works with helical structure). Sven thinks the Polymerase will be large enough to be disturbed by the zincfingers, even if they are coming after the start. An alternative, however, would be to sythesize the promoters in question, Pr and Prm, with the zincfinger binding sites immediately before, after, or in between -35 and -10. The problem with in between is that there is the OR1 region on Pr between -35 and -10 that cannot be changed. Thus, the zincfingers could be adapted to this sequence. There are various possibilites:

  -35      ?       OR1      OR1 & -10
tgttgacT|xxxx|taccTctggcggt|Gataat

possible overlaps/extensions for the 2 binding site (BS) sequencies:

case 1: center overlap, indicated by capital letter: BS1=xxxxtaccT , BS2=Tctggcggt

case 2: upstream overlap with -35, capital letter: BS1=Txxxxtacc , BS2=tctggcggt

case 3: downstream overlap with -10, capital letter: BS1=xxxxtacct , BS2=ctggcggtG

case 4: upstream extension by 1bp, indicated by X: BS1=Xxxxxtacc , BS2=tctggcggt

case 5: downstream extension by 1bp, indicated by X: BS1=xxxxtacct , BS2=ctggcggtX

Alex will now look into that, e.g. trying to put the binding sites just after -10 and possibly with a large overlap, since he is the one most familiar with the literature. Another possibility would be to put it in front, i.e. before -35.