Coding

Part:BBa_K1499200:Design

Designed by: Raman Nelakanti   Group: iGEM14_StanfordBrownSpelman   (2014-08-22)
Revision as of 19:19, 23 October 2014 by Raman2004 (Talk | contribs)

Deinococcus radiodurans UV DNA damage endonuclease - uvsE


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

This part was isolated from the genomic DNA of Deinococcus radiodurans (1). First, genomic DNA was extracted from cells growing in an overnight liquid culture. The gene was simultaneously amplified from genomic DNA and added BioBrick ends using the following protocol: 

Forward primer uvsE (48 bp)

5'-GTTTCTTCGAATTCGCGGCCGCTTCTAGATGACCTCGGCCTGTGAAGC-3'

Reverse primer uvsE (50 bp)

5'-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTAGTCTTTCTTGAACGGCGC-3'

60 ng genomic DNA/reaction 

1 uM primer mix 

25 ul AmplitaQ gold in a total reaction volume of 50 uL 

1 min of 95C denaturation and 45 sec of annealing, x34 cycles


Note 1: The stop codon was modified from the wild-type TGA to the registry's standard TAA using the reverse amplification primer.

Note 2: This coding part does not contain a promoter, RBS, transcriptional terminator, etc. These elements must be added for proper functionality.

Source

Isolated directly from Deinococcus radiodurans genomic DNA, Chromosome 1 [http://www.ncbi.nlm.nih.gov/genome/1020?genome_assembly_id=170301 NCBI Genome]

References

1. White, O et al. (1999) Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1 Science 286(5444):1571-7. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/10567266 10567266]. 2. Earl, AM et al. (2002) Genetic evidence that the uvsE gene product of Deinococcus radiodurans R1 is a UV damage endonuclease. J. Bacteriol. 184(4):1003-9. PMID: [http://www.ncbi.nlm.nih.gov/pubmed/11807060 11807060].