Difference between revisions of "Part:BBa K4361022"
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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 their contribution to <html><a href="https://2022.igem.wiki/tudelft/partnership">our partnership</a></html>, the DTU iGEM 2022 team analyzed the BlcR binding operator with NCBI Blast. In total, they found 25 variants of the sequence originating from different strains of <i>A. tumefaciens</i>, varying in similarity to what we define as the wildtype oligo, [[Part:BBa_K4361001]]. Surprisingly, while they found that all sequences did have conserved nucleotides, these were not specifically for IR1 and IR2 but instead for alternative inverted repeats (see <b>Sequence and Features</b> and <b>Usage and Biology</b> below). From the multiple sequence alignment, two sequences were chosen to be further investigated by us: the consensus sequence formed by the most common nucleotide for each position in the alignment ([[Part:BBa_K4361021]]), and the operator of <i>A. tumefaciens</i> strain Q15, because of its relatively low similarity with the wildtype sequence (this part).
  
 
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Revision as of 05:44, 11 October 2022


BlcR-binding oligo, 49 bp, strain variant

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 their contribution to our partnership, the DTU iGEM 2022 team analyzed the BlcR binding operator with NCBI Blast. In total, they found 25 variants of the sequence originating from different strains of A. tumefaciens, varying in similarity to what we define as the wildtype oligo, Part:BBa_K4361001. Surprisingly, while they found that all sequences did have conserved nucleotides, these were not specifically for IR1 and IR2 but instead for alternative inverted repeats (see Sequence and Features and Usage and Biology below). From the multiple sequence alignment, two sequences were chosen to be further investigated by us: the consensus sequence formed by the most common nucleotide for each position in the alignment (Part:BBa_K4361021), and the operator of A. tumefaciens strain Q15, because of its relatively low similarity with the wildtype sequence (this part).

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.
Figure 3. Results of the third Tapestation experiment, in which the fraction of DNA bound to BlcR was determined for different types of oligos in the presence or absence of 25 μM SSA. The first set of bars represents the results with Part:BBa_K4361000 (scrambled oligo, negative control), the second set of bars corresponds to those of Part:BBa_K4361001 (wildtype oligo, positive control). The third set depicts the measured fraction of bound DNA for this part.