Difference between revisions of "Part:BBa M50041:Design"

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===Design Notes===
 
===Design Notes===
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The second half of our genetic construct was “EColi_Promoter_Cassette,” containing strong RBS, the sequence for green fluorescent protein(GFP) with a terminator immediately following it, an ampicillin resistance marker, and a high copy number origin of replication (ORI). “EColi_Promoter_Cassette” was obtained from Dr. Kara Rogers.
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The second half of our genetic construct was “EColi_Promoter_Cassette,” containing strong RBS, the sequence for green fluorescent protein(GFP) with a terminator immediately following it, an ampicillin resistance marker, and a high copy number origin of replication (ORI). “EColi_Promoter_Cassette” was obtained from Dr. Kara Rogers.<BR>We, in fact, designed 2 constructs, BBa_M50039 and BBa_M50041, where the only difference between them was the cusR-inducible promoter sequence. We explored 2 possible sequences of the cusR-inducible promoter: BBa_M50039’s cusR-inducible promoter sequence was from Zahid et al., 4 and BBa_M50041’s promoter sequence comprised the 250 basepairs preceding the cusR gene and was sourced from NCBI.<BR>We ordered the constructs from DNA 2.0. Notably, we were unable to receive BBa_M50041 exactly as prescribed due to a deletion mutation at the final base, which could cause transcription that is unable to be terminated.
  
  

Revision as of 23:59, 11 December 2016


Positive feedback cusR producer (mutated)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 53
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 53
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 53
    Illegal AgeI site found at 243
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The second half of our genetic construct was “EColi_Promoter_Cassette,” containing strong RBS, the sequence for green fluorescent protein(GFP) with a terminator immediately following it, an ampicillin resistance marker, and a high copy number origin of replication (ORI). “EColi_Promoter_Cassette” was obtained from Dr. Kara Rogers.
We, in fact, designed 2 constructs, BBa_M50039 and BBa_M50041, where the only difference between them was the cusR-inducible promoter sequence. We explored 2 possible sequences of the cusR-inducible promoter: BBa_M50039’s cusR-inducible promoter sequence was from Zahid et al., 4 and BBa_M50041’s promoter sequence comprised the 250 basepairs preceding the cusR gene and was sourced from NCBI.
We ordered the constructs from DNA 2.0. Notably, we were unable to receive BBa_M50041 exactly as prescribed due to a deletion mutation at the final base, which could cause transcription that is unable to be terminated.


Source

http://biocyc.org/gene?orgid=ECOLI&id=G6319

References

“cusR response regulator in two-component regulatory system with cusS [Escherichia coli str. K-12 substr. MG1655”. (2016, Sep. 10). NCBI Reference Sequences.

Zahid N, Zulfiqar S, Shakoori AR. (2012, March). Functional analysis of cus operon promoter of Klebsiella pneumoniae using E. coli lacZ assay. Gene, 495(1):81-88.

Loftin IR, et al. (2005, July). A novel copper-binding fold for the periplasmic copper resistance protein cusF. Biochemistry, 44(31):10533-10540.