Difference between revisions of "Part:BBa K249014"
(3 intermediate revisions by the same user not shown) | |||
Line 10: | Line 10: | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K249014 SequenceAndFeatures</partinfo> | <partinfo>BBa_K249014 SequenceAndFeatures</partinfo> | ||
+ | |||
+ | |||
+ | 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: | ||
+ | |||
+ | |||
+ | [[Image:YFP fluorescence.jpg]] [[Image:bar graph.jpg|400 px]] | ||
+ | |||
+ | 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. | ||
+ | |||
+ | |||
+ | [[Image:YFP denaturing.jpg]] | ||
+ | |||
+ | 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. | ||
Latest revision as of 22:05, 31 October 2009
C-terminal YFP --> Tet inverter
Arabinose repressible Tetracycline inversion of YFP with C-terminal Fusion Arganine Tag for targetting into Lumazine Microcompartment. Has a medium RBS and a double terminator.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.