Difference between revisions of "Part:BBa K249017"
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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. This was observed. | 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. This was observed. | ||
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| + | [[Image:YFP fluorescence.jpg]] | ||
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| + | 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. | ||
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| + | [[Image:YFP denaturing.jpg]] | ||
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| + | Figure 2: 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. | ||
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| + | 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. | ||
Revision as of 17:00, 31 October 2009
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
- 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. This was observed.
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 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.