Difference between revisions of "Part:BBa K2205026"
 
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The Arkin Lab team (2006) produced the promoter probe plasmid (BBa_J61002) for the testing and characterisation of the Anderson promoter library (BBa_J23100 - BBa_J23119). When using this part to characterise the synthetic promoter library produced in this project, many issues concerning expression arose. </p>
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The Arkin Lab team (2006) produced the promoter probe plasmid ([https://parts.igem.org/Part:BBa_J61002 BBa_J61002]) for the testing and characterisation of the Anderson promoter library ([https://parts.igem.org/Part:BBa_J23100 BBa_J23100] - [https://parts.igem.org/Part:BBa_J23119 BBa_J23119]). When using this part to characterise the synthetic promoter library produced in this project, many issues concerning expression arose. </p>
 
<p>
 
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In order to combat these limitations, a new plasmid has been designed to improve the expression of the BBa_J61002 plasmid. Firstly, a bidirectional terminator has been inserted upstream of the iGEM cloning site in order to prevent any leaky expression from the plasmid backbone in which contains a promoter in the reverse direction.</p>
 
In order to combat these limitations, a new plasmid has been designed to improve the expression of the BBa_J61002 plasmid. Firstly, a bidirectional terminator has been inserted upstream of the iGEM cloning site in order to prevent any leaky expression from the plasmid backbone in which contains a promoter in the reverse direction.</p>
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In order to obtain the ability to repress the expression of the promoter being tested, a Lac Operator sequence has been inserted immediately downstream of the promoter insertion site.</p>
 
In order to obtain the ability to repress the expression of the promoter being tested, a Lac Operator sequence has been inserted immediately downstream of the promoter insertion site.</p>
 
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The ribosome binding site BBa_B0034 which is present within the plasmid, is not flanked by any restriction site in order for the swapping out of RBSs. In order to give the plasmid this ability, two restriction sites SacI and BamHI were inserted flanking its sequence.  This is to allow for the RBS to be changed at will if the user wishes to screen a library of RBS as well as promoters.</p>
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The ribosome binding site [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] which is present within the plasmid, is not flanked by any restriction site in order for the swapping out of RBSs. In order to give the plasmid this ability, two restriction sites SacI and BamHI were inserted flanking its sequence.  This is to allow for the RBS to be changed at will if the user wishes to screen a library of RBS as well as promoters.</p>
 
<p>
 
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Finally, the reporter gene currently present in the promoter probe plasmid mRFP, was been replaced with a sfGFP coding sequence. As the sfGFP coding sequence is a significantly shorter than its predecessor, therefore allowing for a faster transcription. This gene sequence also produces a brighter fluorescence than mRFP, with emission wavelengths of 509 nm, interference that can arise when using techniques such as plate readers from high cell density (wavelength of 600 nm) samples become less likely when compared to the emission wavelength of mRFP (584 nm). </p>
 
Finally, the reporter gene currently present in the promoter probe plasmid mRFP, was been replaced with a sfGFP coding sequence. As the sfGFP coding sequence is a significantly shorter than its predecessor, therefore allowing for a faster transcription. This gene sequence also produces a brighter fluorescence than mRFP, with emission wavelengths of 509 nm, interference that can arise when using techniques such as plate readers from high cell density (wavelength of 600 nm) samples become less likely when compared to the emission wavelength of mRFP (584 nm). </p>
  
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[[File:T--Newcastle--Lais--PPP.png|400px|]]
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https://static.igem.org/mediawiki/2017/1/18/T--Newcastle--Lais--PPP.png
  
 
===Characterisation===
 
===Characterisation===

Latest revision as of 19:16, 1 November 2017


pSB1A51 - Promoter Library Screening Plasmid

Description

The Arkin Lab team (2006) produced the promoter probe plasmid (BBa_J61002) for the testing and characterisation of the Anderson promoter library (BBa_J23100 - BBa_J23119). When using this part to characterise the synthetic promoter library produced in this project, many issues concerning expression arose.

In order to combat these limitations, a new plasmid has been designed to improve the expression of the BBa_J61002 plasmid. Firstly, a bidirectional terminator has been inserted upstream of the iGEM cloning site in order to prevent any leaky expression from the plasmid backbone in which contains a promoter in the reverse direction.

In order to obtain the ability to repress the expression of the promoter being tested, a Lac Operator sequence has been inserted immediately downstream of the promoter insertion site.

The ribosome binding site BBa_B0034 which is present within the plasmid, is not flanked by any restriction site in order for the swapping out of RBSs. In order to give the plasmid this ability, two restriction sites SacI and BamHI were inserted flanking its sequence. This is to allow for the RBS to be changed at will if the user wishes to screen a library of RBS as well as promoters.

Finally, the reporter gene currently present in the promoter probe plasmid mRFP, was been replaced with a sfGFP coding sequence. As the sfGFP coding sequence is a significantly shorter than its predecessor, therefore allowing for a faster transcription. This gene sequence also produces a brighter fluorescence than mRFP, with emission wavelengths of 509 nm, interference that can arise when using techniques such as plate readers from high cell density (wavelength of 600 nm) samples become less likely when compared to the emission wavelength of mRFP (584 nm).

T--Newcastle--Lais--PPP.png

Characterisation

To test the functionality of the improved promoter plasmid probe, a sample of the synthetic promoter library produced in this project was assembled into the plasmid using BioBrick assembly. The resulting colonies were miniprepped and insertion was confirmed through sequencing.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 96
    Illegal XbaI site found at 111
    Illegal SpeI site found at 119
    Illegal PstI site found at 979
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 96
    Illegal SpeI site found at 119
    Illegal PstI site found at 979
    Illegal NotI site found at 102
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 96
    Illegal BamHI site found at 173
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 96
    Illegal XbaI site found at 111
    Illegal SpeI site found at 119
    Illegal PstI site found at 979
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 96
    Illegal XbaI site found at 111
    Illegal SpeI site found at 119
    Illegal PstI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2060
    Illegal SapI.rc site found at 191