Difference between revisions of "Part:BBa K4361020"

Revision as of 11:06, 10 October 2022

BlcR-binding oligo, 51 bp, Cro repressor variant

BlcR is a transcription factor originating from the bacterium Agrobacterium tumefaciens (Part:BBa_K4361100). Cro is a repressor protein found in bacteriophage λ. Both proteins form homodimers in vivo to create a dimer with a single DNA-binding domain, though the DNA sequences they each bind differ greatly. As described in Usage and Biology below, one of the core principles of our project was to increase the binding strength between BlcR and DNA. One approach to achieve this was to create a chimera protein which combines the DNA-binding and dimerization domain of Cro with the GHB-recognition and tetramerization domain of BlcR. While its application would be similar to those of the previous parts (Part:BBa_K4361000 through Part:BBa_K4361019), it would require a different binding oligo due to its differing DNA-binding domain.
This part has been designed to test the binding strength between Cro-BlcR and DNA. To create this part, the nucleotides originally designated as belonging to IR1 and IR2 in Part:BBa_K4361001 have been exchanged for the consensus sequence which the Cro repressor binds ('TATCACCgcgGGTGATA', Cro IR1) and its reverse complement ('TATCACCcgcGGTGATA', Cro IR2). As the other nucleotides are identical to those in the wildtype oligo, the BlcR-binding domain of this part thus consists of Cro IR1-tca-Cro IR2, where tca is the original 3 nt linker sequence between IRs. The layout of this oligo presumes that other than the DNA-binding domain, the overall structure of Cro-BlcR is identical to that of wildtype BlcR, including its orientation to the DNA, and it retains its ability to form tetramers when bound to DNA.

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]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4361020 short</partinfo>
-BlcR is a transcription factor originating from the bacterium <i>Agrobacterium tumefaciens</i> ([[Part:BBa_K4361100]]). Cro is a DNA-binding protein found in bacteriophage &lambda;. Both proteins
+BlcR is a transcription factor originating from the bacterium <i>Agrobacterium tumefaciens</i> ([[Part:BBa_K4361100]]). Cro is a repressor protein found in bacteriophage &lambda;. Both proteins form homodimers <i>in vivo</i> to create a dimer with a single DNA-binding domain, though the DNA sequences they each bind differ greatly. As described in <b>Usage and Biology</b> below, one of the core principles of our project was to increase the binding strength between BlcR and DNA. One approach to achieve this was to create a chimera protein which combines the DNA-binding and dimerization domain of Cro with the GHB-recognition and tetramerization domain of BlcR. While its application would be similar to those of the previous parts ([[Part:BBa_K4361000]] through [[Part:BBa_K4361019]]), it would require a different binding oligo due to its differing DNA-binding domain. <br>
+This part has been designed to test the binding strength between Cro-BlcR and DNA. To create this part, the nucleotides originally designated as belonging to IR1 and IR2 in [[Part:BBa_K4361001]] have been exchanged for the consensus sequence which the Cro repressor binds ('TATCACCgcgGGTGATA', Cro IR1) and its reverse complement ('TATCACCcgcGGTGATA', Cro IR2). As the other nucleotides are identical to those in the wildtype oligo, the BlcR-binding domain of this part thus consists of Cro IR1-tca-Cro IR2, where tca is the original 3 nt linker sequence between IRs. The layout of this oligo presumes that other than the DNA-binding domain, the overall structure of Cro-BlcR is identical to that of wildtype BlcR, including its orientation to the DNA, and it retains its ability to form tetramers when bound to DNA.
- 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>
-This part and [[Part:BBa_K4361017]] have been designed to test whether or not multiple separate BlcR tetramers can form on a single DNA molecule. To create this part, a second and third IR1-tca-IR2 set have been added after the 3' end of the original IR2 in the wildtype sequence, each spaced by a 15 nt linker (gcgggtcagcgggtc). The BlcR-binding domain of this part thus consists of IR1-tca-IR2-gcgggtcagcgggtc-IR1-tca-IR2-gcgggtcagcgggtc-IR1-tca-IR2. The distance between the IR sets is assumed to be sufficient to prevent three potential BlcR tetramers from sterically hindering each other, as well as placing them on alternating sides of the DNA molecule due to being spaced 15 nt apart.
-Based off of BBa_K4361001. This oligo was designed for a mutant of the BlcR transcription factor which contained the binding site found in the Cro repressor protein.
 <!-- Add more about the biology of this part here