Part:BBa_K249017

N-terminal YFP --> Tet inverter

Arabinose repressible Tetracycline inversion of YFP with N-terminal Fusion Arganine Tag for targetting into Lumazine Microcompartment. Has a medium RBS and a double terminator.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This part was designed as an inversion device for YFP fluorescence. Upon addition of Arabinose the fluorescence of the protein was expected to drop significantly.

Two constructs (EYFP with either N- or C-terminal 10Arg (10R) tags) were characterized by the following procedure, either in the presence or absence of 0.4% w/v arabinose.

Briefly, 5 mL cultures (LB + 10 mg/mL ampicillin, with or without 0.4 % w/v arabinose) were inoculated with 1 mL of previously prepared culture of E. coli DH5α containing the EYFP constructs. After 1 hour growth at 37 °C (with shaking), 1 mL of the culture was harvested. Cells were centrifuged (13500 g, 5 min) and resuspended in 1 mL of either 8 M urea or buffer TAKM7 (50 mM Tris-Cl pH 7.5 at room temperature, 30 mM NH4Cl, 70 mM KCl, 7 mM MgCl2) containing 1 mg/mL lysozyme. Following a 10 min incubation at room temperature, cell debris was removed by centrifugation (13500 g, 5 min). The fluorescence of the resulting clear cell debris was determined using a Varian Cary Eclipse Fluorescence Spectrophotometer in quartz cuvettes, referenced with respect to the background fluorescence of either 8 M urea or buffer TAKM7. The fluorescence of EYFP was excited at 514 nm; the fluorescence emission was measured through 5 nm slits from 520 to 600 nm. The results are summarized below:

Figure 1: Fluorescence of YFP constructs under native conditions. Fluorescence of buffer TAKM7 (black) and DH5α (grey) cell lysates were used as a control and show no significant fluorescence. Cells expressing C-terminal 10R tagged YFP in the absence (dark yellow) or presence (dark green) of arabinose (N-terminal 10R tagged YFP, light yellow and light green without or with arabinose respectively) showed a significant increase in fluorescence after one hour of growth.

Figure 2. Fluorescence of cell extracts containing R10 YFP constructs.

Figure 3: Fluorescence of YFP constructs under denaturing conditions. Fluorescence of 8 M urea (black) and DH5α (grey) cell lysates were used as a control and show no significant fluorescence. Only cells expressing C-terminal 10R tagged YFP in the absence (dark yellow) of arabinose showed any fluorescence. Cells expressing C-terminal 10R tagged YFP in the presence (dark green) of arabinose (N-terminal 10R tagged YFP, light yellow and light green without or with arabinose respectively) showed no significant fluorescence under denaturing conditions. 8 M urea buffer had a slight fluorescence when excited at 514 nm.

These results indicate that cells express either N- or C-terminal 10R tagged YFP, as shown by an increase in the fluorescence of cleared cell lysates. This fluorescence can be removed by treating cell lysates with 8 M urea, which would denature the protein, reducing the fluorescence of the lysate. This is demonstrated by a change of 10 fluorescence units between total cellular proteins under native conditions compared to denaturing conditions.