Team:Tufts/Design

Design

For this project, we used a strain of Shewanella that had mtrB knocked out. mtrB is responsible for Shewanella's ability to conduct electricity. The idea was to then conjugate the knockout Shewanella with plasmids containing a replacement mtrB, under the control of a promoter that could detect a particular compound. This year, we chose mercury and arabinose. We also initially aimed to include the use of proteolysis tags, which would destroy the mtrB. This way, electricity would not continue to be conducted after thr stimulus was removed.

What are the necessary steps?

  • Conjugate Shewanella with the reporter construct promoters
  • Measure voltage when the stimulus, like mercury or arabinose, is introduced
  • Add in the proteolysis tags and retest the voltage to see if it drops after removal of the stimulus

Mercury

  • We expressed the BBa_K346001 repressor part, designed by Peking University 2010.
  • We used the constitutive promoter, BBa_J23109, since such a level of expression provided Peking the most gradual sensor response to concentration.
  • We also used the mercury inducible promoter by Peking University 2010's igem team to produce mtrB. Specifically, we used the one with mutation three, since it had the most gradual sensor response to concentration.

Arabinose

  • The arabinose assembly (pISara) is based off of pSB1C3, which is provided in the iGEM development kit.
  • The mtrB sequence, as in all other constructs, was obtained from part BBa_K957000, and can be seen above in yellow (the designation of mtrB itself as part K098989 was used internally for reference, this part was not available in the distribution kit)
  • The arabinose inducible promoter and accompanying repressor sequences were obtained from part BBa_K731201, which was used in its entirety, and can be seen upstream of mtrB in the above diagram in both lime green and brown. It was kept under the same constitutive promoter as used in BBa_K731201, in the reverse direction from mtrB.