Part:BBa_K4732001
DDT biosensor (RtER-araC-RFP)
RFP under the control of Rainbow trout estrogen receptor by way of araC: This is our biosensor for xenoestrogens like DDT. In the presence of such molecules, rtER will prevent expression of the araC gene, which in turn relieves repression of RFP. In this case, araC is an inverter module allowing for a red color signal in response to the xenoestrogen.
Characterization of DDT biosensor - Alma 2023
The DDT biosensor works by rtER binding to DDT (which has characteristics similar to estrogen). This causes for the estrogen response element to bond to the receptor stoping transcription of the araC repressor. The araC repressor stops expression of RFP. With no transcription of the araC repressor, RFP can be transcribed and expressed. Tests to determine if the parts of the DDT biosensor worked. Arabinose is a sugar that represses the araC repressor. Due to this, red fluorescence should be seen from the biosensor. Initial tests used 5% arabinose at different volumes added to six different plates, then left over night. The results of the plating are shown in the figure below:
Results showed that arabinose at a 93 uM concentration could stop the araC repressor showing the repressor works. To further this, tests were ran to determine the amount of fluorescence given off by RFP in the lowest concentrations of arabinose that repress araC. The results are shown below:
These results deemed that RFP and araC inverter worked in the circuit. To determine if rtER worked, four liquid cultures were produced. In the first, cells were suspended in LB, 66.6 uM of arabinose and 25 uL of IPTG. This acted as a control. In the second, all the components of the control were added and Estradiol (E2) at 30 uM. In the third, all the components plus 60 uM of E2 were added. In the last, all the components plus 60 uM of E2 were added. These were put into a plate reader and left over night. The results are shown below:
The results showed increased in red fluorescence from adding E2. This indicates that the rtER works and that the circuit works in entirety.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1914
Illegal NheI site found at 1937 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 908
Illegal BamHI site found at 570
Illegal BamHI site found at 3161
Illegal XhoI site found at 464 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 3774
Illegal AgeI site found at 3886 - 1000COMPATIBLE WITH RFC[1000]
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