Part:BBa_K3445001
Estrogen responsive RFP output
This part acts as a estrogen response element controlled inverter. The K123002 piece of the composite part produces the TetR protein, which acts to inhibit the RFP output of the I13521 piece. However, the production of TetR can be blocked by binding of the ERE sequence, therefore allowing the production of RFP.
Initial Characterization (Alma 2020)
This BioBrick is intended to produce TetR in an effort to repress the production of RFP unless acted on by a bound estrogen receptor complex binding to the ERE of the K123002 piece. Therefore, this piece acts as an inverter to elicit a response in the presence of estrogen or estrogen analogs. To characterize the protein expression of this part, we performed SDS PAGE analysis on the overall composite part in comparison to each individual part within it (K123002 and I13521) as well as in comparison to J04450, which constitutively expresses RFP. As RFP and TetR both have a protein weight of approximately 25 kDa (marked on our gel by the red arrow), expression of either (or both) of these proteins will be observed in a similar location.
As can be seen, RFP is being produced by K3445001, which is supported by the red fluorescence seen on the streaked plate, as well as the bar graph showing fluorescence of 3445001 shown below. Additionally, RFP is seen in the I13521 BioBrick in DH5ɑ strain as well as by J04450 in both E. coli strains. However, neither RFP or TetR is observed in the HL2483 strain containing I13521. The lack of RFP is expected considering the part is inhibited by the constitutively produced TetR, but the lack of TetR is slightly surprising. This is likely due to low levels of expression of TetR by HL2483 which cannot be appreciated by this level of separation, meaning the TeT repressible promoter in I13521 is tightly regulated by the presence of TetR. Additionally, no TetR is noted to be produced by the K123002 BioBrick. Therefore, expression of the TetR by K123002 is either at an even lower level than it is in HL2483, meaning it also cannot be analyzed at this level of separation, or the degradation tag attached to this sequence is too powerful and making TetR so unstable it cannot accumulate in the cell. This would have implications on our K3445001 composite part and would be a likely explanation for its red appearance.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 635
Illegal AgeI site found at 747 - 1000COMPATIBLE WITH RFC[1000]
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