Difference between revisions of "Part:BBa K4361014"
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<partinfo>BBa_K4361014 short</partinfo>
 
<partinfo>BBa_K4361014 short</partinfo>
  
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Based off of BBa_K4361001, but inserting the final 5 nucleotides of the 3' end (gcggg) before inverted repeat pair 2.
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BlcR is a transcription factor originating from the bacterium <i>Agrobacterium tumefaciens</i> ([[Part:BBa_K4361100]]). In a homodimer state it contains a single DNA-binding domain that specifically binds one of two DNA sequences. Both sequences are so-called inverted repeat pairs (IRs), short DNA sequences whose ends are reverse complements of each other. For the Blc operator, these sequences are 'ACTCTAATgATTCAAGT' (IR1) and 'ATTAGttgaactCTAAT' (IR2), as further explained in [[Part:BBa_K4361001]]. <br>
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As explained below under <b>Usage and Biology</b>, the length of the linker sequence between IRs (normally 3 nt) is crucial for BlcR's ability to correctly bind the DNA and tetramerize. This part has been designed to show whether or not a larger linker indeed inhibits this behaviour. To create this part, the final five nucleotides of the wildtype oligo (gcggg) have been added between the original linker and IR2. The BlcR-binding domain of this part thus consists of IR1 outer 5-tcagcggg-IR2. As a 3 nt linker is hypothesized to result in two BlcR dimers binding on the same side of a DNA strand, an 8 nt linker as found in this part should result in two dimers binding on opposite parts of the DNA, severely reducing the amount of BlcR bound to DNA strands.  
  
 
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Revision as of 08:18, 10 October 2022


BlcR-binding oligo, 56 bp, IR1 + 5 bp linker + IR2

BlcR is a transcription factor originating from the bacterium Agrobacterium tumefaciens (Part:BBa_K4361100). In a homodimer state it contains a single DNA-binding domain that specifically binds one of two DNA sequences. Both sequences are so-called inverted repeat pairs (IRs), short DNA sequences whose ends are reverse complements of each other. For the Blc operator, these sequences are 'ACTCTAATgATTCAAGT' (IR1) and 'ATTAGttgaactCTAAT' (IR2), as further explained in Part:BBa_K4361001.
As explained below under Usage and Biology, the length of the linker sequence between IRs (normally 3 nt) is crucial for BlcR's ability to correctly bind the DNA and tetramerize. This part has been designed to show whether or not a larger linker indeed inhibits this behaviour. To create this part, the final five nucleotides of the wildtype oligo (gcggg) have been added between the original linker and IR2. The BlcR-binding domain of this part thus consists of IR1 outer 5-tcagcggg-IR2. As a 3 nt linker is hypothesized to result in two BlcR dimers binding on the same side of a DNA strand, an 8 nt linker as found in this part should result in two dimers binding on opposite parts of the DNA, severely reducing the amount of BlcR bound to DNA strands.

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

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Results

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Figure 2. Results of the second Tapestation experiment, in which the fraction of DNA bound to BlcR was determined for different types of oligos. The first bar and bottom dashed line represent the results with Part:BBa_K4361000 (scrambled oligo, negative control), the second bar and top dashed line correspond to those with Part:BBa_K4361001 (wildtype oligo, positive control). The third bar depicts the measured fraction of bound DNA for this part.