Difference between revisions of "Part:BBa K2086000:Design"

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The part had an illegal restriction site in the NapA gene. The NapDA genes and NapBC genes aren't normally adjacent to each other and so were assembled using HiFi assembly.
 
The part had an illegal restriction site in the NapA gene. The NapDA genes and NapBC genes aren't normally adjacent to each other and so were assembled using HiFi assembly.
  
 +
The following primers were used to PCR fragments of the nap operon from E. coli
 +
<p>
 +
NapA(R) -
 +
AATGGCTTACACCTTCTCCAGT</p>
 +
<p>NapA (F) -
 +
CGCGCCGTTGTTGGTCAGCA</p>
 +
<p>NapBC(F) -
 +
TGGAGAAGGTGTAAGCCATTATGAAAAGCCATGACCTGAA</p>
 +
<p>NapBC(R) -
 +
tgcagcggccgctactagtattaAAAACCTGGCTCGACTT</p>
  
 +
<p>The following sequence was ordered from IDT due to necessary removal of restriction enzyme sites that are found in the chromosomal DNA of E. coli and the need to add the BioBrick prefix.</p>
 +
 +
<p>GAATTCGCGGCCGCTTCTAGATGCACACTAACTGGCAAGTTTGCAGCCTGGTCGTGCAGGCCAAAAGCGAACGAATTTCAGACATCAGCACCCAACTGAAC GCCTTTCCCGGCTGTGAAGTTGCTGTCAGCGACGCGCCGAGCGGTCAGTTGATTGTGGTGGTGGAAGCAGAAGACAGCGAAACGCTGATCCAAACCATTGAGT CAGTACGCAACGTAGAGGGCGTGCTGGCGGTGTCGCTGGTTTATCACCAGCAGGAAGAGCAAGGTGAGGAAACACCATGAAACTCAGTCGTCGTAGCTTTATG AAAGCTAACGCCGTTGCGGCGGCTGCGGCGGCT GCCGGTCTCAGCGTGCCGGGCGTTGCCCGCGCCGTTGTTGGTCAGCAGGAAGCCATCAAA</p>
 +
 +
Before HiFi assembly could be completed we realized that the overlap between the fragments was too small in some places and so the following primers were used to lengthen their corresponding fragments and increase the amount of overlap.
 +
 +
 +
<p>NapDA (F) - TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCT</p>
 +
<p>NapDA (R) - TTTGATGGCTTCCTGCTGACCAACAACGG</p>
 +
<p>NapBC  (R) - TTTTTTGCCGGACTGCAGCGGCCGCTA</p>
 +
<p>NapBC(F) - TGGAGAAGGTGTAAGCCATTATGAAAAGCCATGACCTGAA</p>
  
 
===Source===
 
===Source===
  
−
The napDABC gene sequence contains parts derived from the genomic DNA of <i>E. coli</i> (MG1655). Certain sequences were changed and ordered from IDT in order to eliminate illegal restriction sites.
+
The napDABC gene sequence contains sections of the nap operon derived from the genomic DNA of <i>E. coli</i> (MG1655).
  
 
===References===
 
===References===

Latest revision as of 21:43, 19 October 2016


napDABC (Periplasmic Nitrate Reductase)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1991
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 479
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 324
    Illegal SapI.rc site found at 238
    Illegal SapI.rc site found at 1734


Design Notes

The part had an illegal restriction site in the NapA gene. The NapDA genes and NapBC genes aren't normally adjacent to each other and so were assembled using HiFi assembly.

The following primers were used to PCR fragments of the nap operon from E. coli

NapA(R) - AATGGCTTACACCTTCTCCAGT

NapA (F) - CGCGCCGTTGTTGGTCAGCA

NapBC(F) - TGGAGAAGGTGTAAGCCATTATGAAAAGCCATGACCTGAA

NapBC(R) - tgcagcggccgctactagtattaAAAACCTGGCTCGACTT

The following sequence was ordered from IDT due to necessary removal of restriction enzyme sites that are found in the chromosomal DNA of E. coli and the need to add the BioBrick prefix.

GAATTCGCGGCCGCTTCTAGATGCACACTAACTGGCAAGTTTGCAGCCTGGTCGTGCAGGCCAAAAGCGAACGAATTTCAGACATCAGCACCCAACTGAAC GCCTTTCCCGGCTGTGAAGTTGCTGTCAGCGACGCGCCGAGCGGTCAGTTGATTGTGGTGGTGGAAGCAGAAGACAGCGAAACGCTGATCCAAACCATTGAGT CAGTACGCAACGTAGAGGGCGTGCTGGCGGTGTCGCTGGTTTATCACCAGCAGGAAGAGCAAGGTGAGGAAACACCATGAAACTCAGTCGTCGTAGCTTTATG AAAGCTAACGCCGTTGCGGCGGCTGCGGCGGCT GCCGGTCTCAGCGTGCCGGGCGTTGCCCGCGCCGTTGTTGGTCAGCAGGAAGCCATCAAA

Before HiFi assembly could be completed we realized that the overlap between the fragments was too small in some places and so the following primers were used to lengthen their corresponding fragments and increase the amount of overlap.


NapDA (F) - TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCT

NapDA (R) - TTTGATGGCTTCCTGCTGACCAACAACGG

NapBC (R) - TTTTTTGCCGGACTGCAGCGGCCGCTA

NapBC(F) - TGGAGAAGGTGTAAGCCATTATGAAAAGCCATGACCTGAA

Source

The napDABC gene sequence contains sections of the nap operon derived from the genomic DNA of E. coli (MG1655).

References