Part:BBa_K4166013

DDT biosensor (RtER-TetR-RFP)

RFP under the control of Rainbow trout estrogen receptor by way of TetR: This is our biosensor for xenoestrogens like DDT. In the presence of such molecules, rtER will prevent expression of the TetR gene, which in turn relieves repression of RFP. In this case, TetR is an inverter module allowing for a red color signal in response to the xenoestrogen

Characterization of DDT Biosensor - Alma 2023

This biosensor should produce a red signal in response to estrogen or xenoestrogens. We tested this ability using Estradiol. Our protocol was as follows: cells were grown to a mid-log OD, induced with IPTG (for expression of the estrogen receptor), and incubated with varying concentrations of estradiol in a 96 well plate. RFP production was monitored by fluorescence (585nm excitation, 625nm emission) and growth was monitored by OD at 600nm. Below is the fluorescence/OD of different strains over time. This represents replicate measurements across several independent experiments:

There was a statistically significant increase in RFP production in the presence of estradiol (E2) compared to a DMSO control. Furthermore, tests were ran to determine the lowest concentration of E2 needed for RFP expression. Cells were grown in a liquid culture containing LB for one day. From there, 25 uL of IPTG was added to each. Each liquid culture contained a different concentration of E2 (6.7 uM, 10 uM, 20 uM, 40 uM). These were incubated in a 96 well plate. RFP production was monitored by fluorescence (585nm excitation, 625nm emission). The results are shown below:

The results showed some randomness as red fluorescence at 10 uM was lower than at 6.7 uM and 40 uM was lower than 20 uM. However, the graph does show significant RFP production in estradiol.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1922
  Illegal NheI site found at 1945
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 908
  Illegal BamHI site found at 570
  Illegal XhoI site found at 464
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 3305
  Illegal AgeI site found at 3417
  
• 1000
  COMPATIBLE WITH RFC[1000]