Part:BBa_K577895

TetR repressed RFP

A composite part containing the TetR gene, C0040, turned on by a constitutive promoter, I14033. The TetR represses the pTet promoter in front of the RFP gene. By addition of tetracycline or aTc, this repression is inhibited and the cells should fluoresce red.

Characterization by iGEM TU_Darmstadt 2019

When transforming E. coli BL21 (DE3) using BBa_K577895 the transformed culture expressed strong red color even without the addition of AHT. The expression of the tetR repressor seems to be too weak to properly inhibit the expression of the upstream following mRFP1 (Fig. 1).

Figure 1: BBa_K577895 transformed in E. coli BL21 (DE3) without the addition of anhydrotetracycline after cultivation overnight. The culture expresses strong red color.

The expression of mRFP1 was measured using a SpectraMax M5E (Ex: 583 nm, Em: 607 nm) over a period of six hours with and without the addition of 200 µg/L AHT. The results are shown in Fig. 2.

Figure 2: Expression of mRFP1 (E. coli BL21 DE3) with and without the addition of anhydrotetracycline over a period of 6 hours. 0 h corresponds to the time of induction with 200 µg/L AHT at an OD600 of 0.1.

The addition of 200 µg/L AHT does not seem to have an effect on the expression of mRFP1. With and without the addition of AHT the expression of mRFP1 increases over the monitored time period.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1529
  Illegal AgeI site found at 1641
  
• 1000
  COMPATIBLE WITH RFC[1000]