Difference between revisions of "Part:BBa B0040:Design"

(→‎Design Notes)
(→‎Design Notes)
 
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The size of the spacer was chosen to meet the minimum length of a sequence      that can be queried using the BLAST search engine. However, subsequences of      it can be used to design shorter spacers. The sequence was selected from many      more sequences randomly generated using the <a href="http://www.lifesci.ucsb.edu/~maduro/random.htm"> Random      DNA Generator </a> engine; the GC% parameter used as input was 50%. The sequences      were selected based on the following constraints listed in their order of      importance: the absence of any putative promoter regions, a low degree of      homology with the Elowitz plasmid (whose components are widely used in our      designs), no homology with other <em>E.coli</em> sequences as shown by BLASTN      search results and the presence of a number of TAA stop codons. The second      constraint was the most stringent leading to the elimination of most sequences.     
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The size of the spacer was chosen to meet the minimum length of a sequence      that can be queried using the BLAST search engine. However, subsequences of      it can be used to design shorter spacers. The sequence was selected from many      more sequences randomly generated using the [http://www.lifesci.ucsb.edu/~maduro/random.htm Random      DNA Generator] engine; the GC% parameter used as input was 50%. The sequences      were selected based on the following constraints listed in their order of      importance: (1) the absence of any putative promoter regions, (2) a low degree of      homology with the Elowitz plasmid (whose components are widely used in our      designs), (3) no homology with other <em>E.coli</em> sequences as shown by BLASTN      search results and (4) the presence of a number of TAA stop codons. The second      constraint was the most stringent, leading to the elimination of most candidate sequences.     
  
 
DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA.  Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur):
 
DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA.  Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur):
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Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences.
 
Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences.
  
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In the process of selecting the best sequence it appeared that a good alternative      sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA.      The sequence contains no putative promoters and shows moderate homology with      the 5' end of the Ampicillin resistance gene. However a strong promoter sequence      starts 12 bp downstream of this sequence, and therefore the sequence presented      above was preferred.  
+
In the process of selecting the best sequence it appeared that a good alternative      sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA.      The sequence contains no putative promoters and shows moderate homology with      the 5' end of the Ampicillin resistance gene. However, a strong promoter sequence      starts 12 bp downstream of this sequence, and therefore the sequence presented      above was preferred.  
  
 
The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.
 
The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.

Latest revision as of 19:32, 12 April 2007


Spacer.1 (generic)


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]


Design Notes

The size of the spacer was chosen to meet the minimum length of a sequence that can be queried using the BLAST search engine. However, subsequences of it can be used to design shorter spacers. The sequence was selected from many more sequences randomly generated using the [http://www.lifesci.ucsb.edu/~maduro/random.htm Random DNA Generator] engine; the GC% parameter used as input was 50%. The sequences were selected based on the following constraints listed in their order of importance: (1) the absence of any putative promoter regions, (2) a low degree of homology with the Elowitz plasmid (whose components are widely used in our designs), (3) no homology with other E.coli sequences as shown by BLASTN search results and (4) the presence of a number of TAA stop codons. The second constraint was the most stringent, leading to the elimination of most candidate sequences.

DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA. Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur):

T->A @ 85

T->A @ 42

C->T @ 79

A->T @ 64

A->T @ 31

T->A @ 34

C->A @ 37

Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences.

In the process of selecting the best sequence it appeared that a good alternative sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA. The sequence contains no putative promoters and shows moderate homology with the 5' end of the Ampicillin resistance gene. However, a strong promoter sequence starts 12 bp downstream of this sequence, and therefore the sequence presented above was preferred.

The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.

Source

Randomly generated and optimized for several parameters (see Design notes).

References