Part:BBa_K2758000

Mercury activated Killer Red Protein suicide switch

The Killer Red Construct is a double condition suicide sequence. The first condition is the presence of mercury. When the bacteria are in an environment containing mercury, the production of RBS (BBa_B0030) is initiated through the Promotor (BBa_K346002). Then, the Killer Red protein (BBa_K1184000) is produced. The second condition is the presence of light. In order for the Killer red to activate, light (540-585 nm) needs to be shined onto the bacteria to initiate the reaction.

Usage and Biology

This part is designed to work in conjunction with a mercury accumulator device, specifically part BBa_K1355003 designed by the iGEM14_UFAM_BRAZIL team.

We chose to use BBa_B0030 as a moderate binding RBS in order to allow a delay in the expression of Killer Red protein in relation to the accumulator.

Evidence for Bacteria Transformed with Killer Red Construct

The gel contains the restriction products of the plasmid using the HindIII (Well 5) and BamHI (Well 6) enzymes. Both restrictions have the same size, showing that both restrictions were successful and that the two plasmids are the same. It is also evidence that the HindIII restriction site we added into our construct is present, and it was cut by the enzyme, further suggesting that the construct was ligated to the plasmid backbone. Once we incubated three tubes of bacteria for 60 minutes under normal conditions (37 degrees celsius), we added 0 ppm Mercury (Hg), 25 ppm Hg, and 50 ppm Hg to each tube respectively. This chart is representing the time of absorption of mercury for different amounts of mercury. The bacteria in this chart has been transformed with Killer Red. As time goes on, each of the samples gets absorbed, with the 0ppm sample taking the shortest amount of time and 50 ppm taking the least amount of time. Once we incubated three tubes of bacteria for 60 minutes under normal conditions (37 degrees celsius), we added 0 ppm Mercury (Hg), 25 ppm Hg, 50 ppm Hg, and 100 ppm Hg to each tube respectively. This chart is representing the time of absorption of mercury for different amounts of mercury. The bacteria in this chart has been transformed with the accumulator. As time goes on, each of the samples gets absorbed, with the 0ppm sample taking the shortest amount of time and 100ppm taking the least amount of time. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 237
  Illegal BsaI.rc site found at 528