Difference between revisions of "Part:BBa K731700"
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<partinfo>BBa_K731700 short</partinfo>
 
<partinfo>BBa_K731700 short</partinfo>
  
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We used this part to test terminators: the two subsequent proteins allow termination efficiency's quantification as the ratio between Venus's and Cherry's fluorescence.
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This part is used to analyse terminators: two subsequent fluorescent proteins (mCherry and A206K Venus), separated by Prefix-Suffix linker, allow standardization of terminator's presence effect. The vector is a modified pET21b; it contains Ampicillin resistance gene and is inducible by IPTG (T7promoter-lacO).
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The vector is pET21b, that contains lacI, t7 promoter and ampicillin resistance gene.We inserted the prefix-suffix linker between the two proteins, to allow terminator's characterization, and mutated the two illegal sites present in the original plasmid.
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The two fluorescent proteins have partially overlapping emission spectra, thus, to avoid unwanted interferences, the fluorimetric measures should be taken with these parameters:
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The combined use of [[Part:BBa_K731710]] and BBa_K731700 allow also to analyse any potential difference in terminators' activity due to different polymerases.
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Venus excitation: 485nm, Venus emission: 528nm
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Cherry excitation: 528nm, Cherry emission: 615nm.
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This is the twin construct of BBa_K731710, where the T7 promoter has been replaced with Ptac. The two constructs allow terminators' characterization with different RNApolymerase and analysis of any potential difference.
 
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We tested both constructs in the E.Coli strain BL21pLysS, that has both Coli and T7 polymerases.
 
  
 
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Revision as of 08:09, 24 August 2012

Platform for terminators analysis under the control of T7 promoter

This part is used to analyse terminators: two subsequent fluorescent proteins (mCherry and A206K Venus), separated by Prefix-Suffix linker, allow standardization of terminator's presence effect. The vector is a modified pET21b; it contains Ampicillin resistance gene and is inducible by IPTG (T7promoter-lacO).

The combined use of Part:BBa_K731710 and BBa_K731700 allow also to analyse any potential difference in terminators' activity due to different polymerases.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 6850
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 6856
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 6850
    Illegal BglII site found at 6011
    Illegal BamHI site found at 6832
    Illegal XhoI site found at 753
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 6850
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 6850
    Plasmid lacks a suffix.
    Illegal XbaI site found at 6865
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 714
    Illegal NgoMIV site found at 1051
    Illegal NgoMIV site found at 4231
    Illegal NgoMIV site found at 4391
    Illegal NgoMIV site found at 5979
    Illegal AgeI site found at 6800
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 2228
    Illegal SapI.rc site found at 3310