Difference between revisions of "Part:BBa K4361008"
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<partinfo>BBa_K4361008 short</partinfo>
 
<partinfo>BBa_K4361008 short</partinfo>
  
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Similar to BBa_K4361007, but here incorporating the perfect inverted repeat found in BBa_K4361004.
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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 described in [[Part:BBa_K4361004]] and [[Part:BBa_K4361005]], the original sequence of IR1 is not a perfect reverse complement of itself. Furthermore, as described in [[Part:BBa_K4361007]], only the outer 5 nucleotides of IR2 are reverse complementary instead of the outer 8 in IR1, meaning IR1 can be changed to also only have its outer 5 nucleotides be reverse complementary. This part has been designed to combine the changes in [[Part:BBa_K4361004]] and [[Part:BBa_K4361007]], meaning nucleotides 6-12 of IR1 RV 1 have been replaced by those of IR2, resulting in 'ACTCTttgaactAGAGT' (IR1 RV 1 outer 5).
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In the original binding sequence, only the outer 5 nucleotides on each end of IR2 form the inverted repeat, whereas the outer 8 nucleotides form the repeat in IR1. This part has been designed to test whether or not shortening the complementary sequences on each end of IR1 would increase the binding strength between BlcR and DNA. To create this part, nucleotides 6-12 of IR1 have been replaced by those of IR2, resulting in 'ACTCTttgaactCAAGT' (IR1 outer 5). The BlcR-binding domain of this part thus consists of IR1 RV 1 outer 5-tca-IR2, where tca is the original 3 nt linker sequence between IRs.
  
 
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Revision as of 07:49, 10 October 2022


BlcR-binding oligo, 51 bp, IR1 perfect RV 1 outer 5 + 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 described in Part:BBa_K4361004 and Part:BBa_K4361005, the original sequence of IR1 is not a perfect reverse complement of itself. Furthermore, as described in Part:BBa_K4361007, only the outer 5 nucleotides of IR2 are reverse complementary instead of the outer 8 in IR1, meaning IR1 can be changed to also only have its outer 5 nucleotides be reverse complementary. This part has been designed to combine the changes in Part:BBa_K4361004 and Part:BBa_K4361007, meaning nucleotides 6-12 of IR1 RV 1 have been replaced by those of IR2, resulting in 'ACTCTttgaactAGAGT' (IR1 RV 1 outer 5). In the original binding sequence, only the outer 5 nucleotides on each end of IR2 form the inverted repeat, whereas the outer 8 nucleotides form the repeat in IR1. This part has been designed to test whether or not shortening the complementary sequences on each end of IR1 would increase the binding strength between BlcR and DNA. To create this part, nucleotides 6-12 of IR1 have been replaced by those of IR2, resulting in 'ACTCTttgaactCAAGT' (IR1 outer 5). The BlcR-binding domain of this part thus consists of IR1 RV 1 outer 5-tca-IR2, where tca is the original 3 nt linker sequence between IRs.

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.