Part:BBa_K2450301

pTet promoter coding eYFP

A pTet promoter and RBS encoding for YFP.

This part was designed to test the efficacy of a modified TetR (Tet repressor), BBa_K2450201. It is a medium strength pTet promotor.

This part will work as a reporter for a system relying on a change in the levels of TetR repression.

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]

Usage and Biology

pTet is the promoter that can be bound by the Tet repressor, a dimer that is prevented from binding DNA by the presence of the antimicrobial tetracycline (ATC). TetR is a very tight-binding repressor, meaning that leakage is unlikely (in contrast to a system such as Lac).

For more information on how this part was used in our project, please see our wiki: http://2017.igem.org/Team:Oxford

We have demonstrated that our part works as participated, by plate reader experiments measuring the amount of YFP fluorescence produced over time in different conditions. The results are detailed below.

Characterisation

This part required a two-step characterisation:

• Check TetR can bind to pTet and repress eYFP production
• Check ATC can relieve the repression by TetR (link to TetR design page) and hence prove that TetR is the component that is causing the repression

Two strains of E. coli were used to test our part:

1. DH5a containing pTet-eYFP plasmid
2. JBEI-2492 - a strain that contains TetR in its bacterial genome transformed with pTet-eYFP plasmid

We did an initial fluorescence microscopy test with the pTet-eYFP to see if YFP was being produced constitutively.

Figure 1: Initial microscopy images

Figure 2: Steady State Analysis of pTet sensitivity to TetR

Figure 3: Steady State Analysis of TetR sensitivity to ATC in pTet+eYFP

We ran steady state analyses of the two strains as we were interested in the qualitative ability of TetR to repress pTet.

To achieve this, we performed the following steps:

• Three colonies of each strain were picked and grown overnight in minimal media (M9-clear liquid, less effect on fluorescence readings in the plate reader than LB)
• The OD600 of the cells was measured to ensure growth that reached stationary phase.
• The cells were diluted and fluorescence for YFP was recorded in a BMG Labtech Clariostar Plate reader at 495 +/– 15nm, as well as the absorbance at 600nm
• Three aliquots of the overnight culture of each colony of the strains were taken and diluted down.
• The aliquots were introduced to various levels of ATC and allowed to grow until stationary phase was reached.
• The readings for three technical repeats of the three biological repeats were taken on the plate reader.

As you can see by the graph in Figure 2; the significant (nearly 4-fold) difference in Fluorescence/Absorbance with the introduction of TetR suggests that our pTet is sensitive to repression by TetR.

As shown in Figure 3, ATC has been used to determine that repression is indeed being caused by TetR and to mimic cruzipain.

Repression is not significantly relieved when 1nM of ATC is added. This helps show that our system has been optimised for low false positives, as very low concentrations of ATC do not result in a large release of repression.

However at 10nM, the Fluorescence/Absorbance does increase approximately 2-fold. This further clarifies that TetR is the species within the cell that is repressing the production of eYFP, as it has a known sensitivity to ATC.