Difference between revisions of "Part:BBa K494001"

(New page: In general we want to provide a new principle of gene regulation which can be further developed, tested and optimizted by everybody. Therefore we focus on providing the parts needed for ve...)
 
 
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In general we want to provide a new principle of gene regulation which can be further developed, tested and optimizted by everybody. Therefore we focus on providing the parts needed for verification and testing of new individual switches. We provide a plasmid which can be used for further cloning, a positive control to test the general functionality and the constructs we characterized for comparison.
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__NOTOC__
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[edit] Screening system: Backbone BBa_K494001
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<partinfo>BBa_K494001 short</partinfo>
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<br><br>
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This improved screening system, based on the pSB1A10 plasmid, is optimized for the evaluation of PoPS-based devices. With this BioBrick we provide a backbone which allows further cloning to test individual switches which can be easily designed using the principles described in the [http://2010.igem.org/Team:TU_Munich/Project TU Munich iGEM 2010 wiki].
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<br><br>
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The backbone and general construction scheme is based on the <partinfo>pSB1A10</partinfo> plasmid, and like its precursor it is designed to carry two fluorescent proteins, one within the biobrick cloning site. We substituted RFP for mCherry, which combines fast maturation times with acceptable quantum yields and fluoresces red. RFP was removed from the backbone using restriction enzymes SgrA1 and Pst1 and a short linker was inserted. In the submitted version of this Backbone, mCherry was introduced into the standard BioBrick cloning site using EcoRI and PstI. <br><br>
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This brings two major advantages: First of all the second fluorescent protein can be exchanged to any other reporter protein in only one cloning step, which makes BBa K494001 a valueable backbone for many applications. Variation of the reporter proteins allows easy adjustment to measuring equipment and methods beyond fluorescence measurements can be applied. Since GFP stays as an internal control, experimental settings and induction strength can still be estimated. <br><br>
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Another major advantage is the possibility to clone two BioBricks next to mCherry into the plasmid. The E/X site in front of mCherry can be used to insert terminators, promoters and other PoPS devices which could previously be tested using <partinfo>pSB1A10</partinfo>. We would recommend adding an additional promoter in front of the analyzed part in this case since our measurements showed only weak RFP expression using pSB1A10.<br>
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After the mCherry protein, the S/P site can now be utilized for insertion of another DNA encoded part. For example K494003-K494006 are based on K494001, with <partinfo>K494003</partinfo> and <partinfo>K494005</partinfo> carrying only a terminator in between GFP and mCherry while <partinfo>K494004</partinfo> and <partinfo>K494006</partinfo> are also equipped with an inducible signal which, in theory, should allow control over the terminator. Together with <partinfo>K494002</partinfo> which serves as a positive control with only the additional pBAD promoter in front of mCherry, K494001 allows to build up a complete setup for evaluation of terminator efficiency and switching elements.  
  
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We designed a new screening systems based on the non-functional pSB1A10 plasmid. We improved its features for ‘’in vivo’’ characterization of PoPS-based devices using fluorescent reporters . The plasmid still contains the Pbad arabinose-inducible induction system as a tunable input and eGFP as an internal standard for induction. However, we altered the reporter protein to mCherry. Furthermore we adjusted the BioBrick cloning site to allow cloning of additional parts independent from the Input/Output measurement. This screening plasmid is designed to be used with a second Arabinose inducible promoter BBa_I13453 which is not included in this part.
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For spectra of the GFP and mCherry fluorescence of this system, see <partinfo>K494002</partinfo> (positive control).
  
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The improved screening system is optimized for the evaluation of PoPS-based devices in fluorescence measurements. RFP which was known to contain an RNase restriction site was replaced by mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. A unique challenge for the characterization of our switches is the expression of a corresponding signal independent of the input/output measurement. Thus, we moved the BioBrick cloning site resulting in the fluorescent reporter being inside the cloning site and giving the possibility to clone independent parts behind the reporter protein. To fully function our screening plasmid need the arabinose inducible promoter BBa_I13453 in front of the PoPS-based device to screen. Using a second arabinose inducible promoter, we were able to keep eGFP as an internal standard for the tunable input via the Pbad arabinose-inducible induction system. The two identical promoters ensure the same rate of induction for eGFP and the tested PoPS-based device. Thus, obtaining comparable screening results is easy. Unfortunately, this design implicates a minor disadvantage. Two cloning steps are needed to gain an functional construct for testing any PoPS-based device.
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K494001 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K494001 parameters</partinfo>
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<!-- -->

Latest revision as of 03:14, 28 October 2010

Superior Screening Plasmid

This improved screening system, based on the pSB1A10 plasmid, is optimized for the evaluation of PoPS-based devices. With this BioBrick we provide a backbone which allows further cloning to test individual switches which can be easily designed using the principles described in the [http://2010.igem.org/Team:TU_Munich/Project TU Munich iGEM 2010 wiki].

The backbone and general construction scheme is based on the pSB1A10 plasmid, and like its precursor it is designed to carry two fluorescent proteins, one within the biobrick cloning site. We substituted RFP for mCherry, which combines fast maturation times with acceptable quantum yields and fluoresces red. RFP was removed from the backbone using restriction enzymes SgrA1 and Pst1 and a short linker was inserted. In the submitted version of this Backbone, mCherry was introduced into the standard BioBrick cloning site using EcoRI and PstI.

This brings two major advantages: First of all the second fluorescent protein can be exchanged to any other reporter protein in only one cloning step, which makes BBa K494001 a valueable backbone for many applications. Variation of the reporter proteins allows easy adjustment to measuring equipment and methods beyond fluorescence measurements can be applied. Since GFP stays as an internal control, experimental settings and induction strength can still be estimated.

Another major advantage is the possibility to clone two BioBricks next to mCherry into the plasmid. The E/X site in front of mCherry can be used to insert terminators, promoters and other PoPS devices which could previously be tested using pSB1A10. We would recommend adding an additional promoter in front of the analyzed part in this case since our measurements showed only weak RFP expression using pSB1A10.
After the mCherry protein, the S/P site can now be utilized for insertion of another DNA encoded part. For example K494003-K494006 are based on K494001, with BBa_K494003 and BBa_K494005 carrying only a terminator in between GFP and mCherry while BBa_K494004 and BBa_K494006 are also equipped with an inducible signal which, in theory, should allow control over the terminator. Together with BBa_K494002 which serves as a positive control with only the additional pBAD promoter in front of mCherry, K494001 allows to build up a complete setup for evaluation of terminator efficiency and switching elements.

For spectra of the GFP and mCherry fluorescence of this system, see BBa_K494002 (positive control).

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 4495
    Illegal NheI site found at 3696
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 4501
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4495
    Illegal BamHI site found at 3635
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 4495
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 4495
    Plasmid lacks a suffix.
    Illegal XbaI site found at 4510
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 39
    Illegal AgeI site found at 3470
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 1450
    Illegal BsaI.rc site found at 4371
    Illegal SapI site found at 3452