Part:BBa_K2135003

petF on pTet inducible promoter

petF, which encodes for ferredoxin, is put on a TetR-pTet inducible promoter. Various concentrations of aTc (anhydrotetracycline; a tetracycline analog) will result in respective amount of petF transcription. Below, you can see how the [http://2016.igem.org/Team:WashU_StLouis WashU_STL 2016 team] used this part to produce increasing amount of ferredoxin.

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
  COMPATIBLE WITH RFC[1000]

Uses

In 2016, the [http://2016.igem.org/Team:WashU_StLouis WashU_STL team] wanted to overexpress electron donors in e. coli in order to create a suitable environment for nitrogenase activity in nondiazotrophic bacteria. Nitrogenase fixes atmospheric nitrogen gas and converts it to useful nitrates that the cell needs. It requires reduced electron donors to function properly. In many diastrophic cyanobacteria, ferredoxin (Fd) reduces nitrogenase to activate it[1]. The WashU team used this part to overproduce ferredoxin in order to increase the amount of reduced ferredoxin available. In order to measure the amount of electron donors, the team used a biotin assay. Biotin is a biologically produced vitamin that is involved in fatty acid synthesis. Biotin biosynthesis is dependent on a reducing agent, which, in this case, is ferredoxin[2]. Below is a graph indicating the amount of biotin produced versus the concentration of aTc added to activate pTet to transcribe petF.

Here is a diagram for how aTc can activate this part. The first picture shows how pTet is inhbited by TetR. The second photo shows, when aTc is added, it can inhibit the inhibitor, thereby freeing pTet for transcription of the desired gene (petF in this example). Depending on the concentration of aTc added, not all plasmids will be uninhibited (as seen below), resulting in various levels of activation depending on the amount of aTc added. In other words, aTc is the limiting reagent in the production of the desired gene product (ferredoxin).

References

1. Rees, Douglas C., and James B. Howard. "Nitrogenase: standing at the crossroads." Current opinion in chemical biology 4.5 (2000): 559-566
2. Lin, Steven, and John E. Cronan. "Closing in on complete pathways of biotin biosynthesis." Molecular BioSystems 7.6 (2011): 1811-1821.