Part:BBa_K4255017

This is a naringenin detector

This part shows GFP fluorescence when naringenin is present in the bacterial culture.

Usage and Biology

At this year's iGEM competition, we developed synthetic pathways for delphinidin, one particular kind of anthocyanin. Seven enzymes are needed to produce delphinidin. One of the intermediates is named naringenin, a common plant extract substance. All intermediates, including the naringenin, are colorless and onerous to observe using a simple and on-the-spot method. Therefore, a working biosensor that can report fluorescence upon detecting the naringenin would be helpful.

In 2014, iGEM team TU_Darmstadt used to developed a naringenin sensor, K1497020

The sensor will produce fluorescence upon receiving naringenin signal, but unfortunately, after we synthesized this naringenin detector and constructed it on the PETDuet-1 plasmid, we failed to see GFP fluorescence signal even using 50 μM standard naringenin. PETDuet-GFP was used as a control to show at the right panel. We closely studied their sequence and referred to the original research paper. We realized their results were correct and great, but they made a typo mistake when uploading the sequence to the server. The GFP sequence should be on the opposite side of the activator protein in principle, but in the released sequence, the GFP and the FdeR regulator are on the same side. This will silence the GFP because the FdeR will only activate genes in opposite directions.

Fig. 1: The working principle of a naringenin sensor using FdeR protein as the regulator.

Fig. 2: The previously upload FdeR regulator does not produce GFP signal upon adding naringenin 50uM.

Fig. 3: Our construction of the new expression system.

Fig. 4: Expression of GFP verified by SDS-Page with the new naringenin sensor and FdeR regulator.

The result showed upon introduction of naringenin into the bacteria culture, the sensor will successfully express GFP proteins at the expected band. However, our experiments are limited in that the GFP is not bright enough. We think either it is our GFP is not correctly expressed, or the FdeR protein is not sufficiently produced to give a strong GFP expression.

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]