Part:BBa_K1343000

P_tet->NheI->RBS->luxI->double terminator

This part generates AHL (Source organism: V. fischeri). The promoter is P_tet which induced by Tetracycline or by its analog anhydrotetracycline (aTc). Before the promoter there is the prefix site and after the promoter there is a restriction site of NheI, so the promoter can be replaced by a restriction enzyme reaction. In addition, after the NheI restriction site, there is an RBS and luxI which is coding to AHL. At the end of the part, there is double terminator and the Suffix site.

We have colaborate with the BGU team. They conducted an AHL detection experiment (Marks R.S.and Kushmaro A., 2011) to help us test this part.

Positive control: For a positive control the indicator strain, CV026, was incubated with a synthetic AHL (3-OXO-C6). When this strain senses AHL it changes color to purple. A plate was covered with soft agar containing the indicator strain, and then synthetic AHL was added. After incubation the plates were purple as a response for the high levels of the AHL.

Picture 1. Positive control- soft agar containing the detector strain CV026 after incubation with synthetic AHL.

TOP10 bacteria containing gate 1The TOP10 bacteria containing Gate 1 were spread on LB agar plate and after half an hour the plate was covered by soft agar containing the indicator strain CV026. After incubation, a purple color appeared on the plates. Although aTc wasn't added to the bacteria, the leakiness of the Ptet promoter enabled sufficient expression of AHL. The results means gate 1 was assembled correctly and functions as expected.

Picture 2. TOP10 bacteria with gate 1 on LB agar plate which was covered by a soft agar containing the indicator strain.

In a different assay a soft agar with the indicator strain was spread on the plates. 100µl of the supernatant from the TOP10 growth medium were spread on top of the soft agar. After incubation, a color change didn't appear. This time the low expression of AHL caused by the leakiness of the Ptet promoter wasn't sufficient in the supernatant in order to change the color of the indicator strain. The reason for the results is that the induction of gate 1 by aTc was missing.

Picture 3. 100µl of the supernatant from the TOP10 containing gate 1 growth medium on soft agar with the indicator strain.

Link to the experiment on Technion Wiki page: http://2014.igem.org/Team:Technion-Israel/Experiments#gate1

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 64
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]