Difference between revisions of "Part:BBa K3945014:Design"

 
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===Design Notes===
 
===Design Notes===
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<p> <b>Quick Screening:</b> Screening for positive colonies following cloning is often a long and tedious task that often requires screening of many colonies. With the help of a unique feature on the Xpress vector, screening for a positive colony could be done in one single step. Xpress contains two selection markers: a kanamycin resistance gene and an ampicillin resistance gene. However, upon cloning of the insert into the appropriate spot in the multiple cloning sites, the kanamycin resistance gene will be knocked out. As such, successfully cloned colonies will only be resistant to ampicillin whereas colonies that have not been engineered properly will retain their kanamycin resistance. By streaking colonies on ampicillin and kanamycin plates, successful and unsuccessful colonies could be easily differentiated from one another as the successful colonies are expected to only grow on the ampicillin plates while all other negative colonies will show growth on both kanamycin and ampicillin plate. Thus, in this one simple step Xpress allows for fast screening of positive colonies. </p>
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<p> <b>Ease of Expression:</b> <I>E. coli</i> has been an extremely powerful factory for producing proteins. Heterologous protein expression, however, could sometimes prove challenging. In particular, for proteins that are not suited to the natural expression conditions found in E. coli or for insoluble proteins that are difficult to produce. Xpress contains a GST tag which is a protein that is highly soluble and easily produced by <I>E. coli</i>. fusion of GST to heterologous proteins has proven to help with their expression as GST can act as a chaperone that helps with the proper folding of the protein [1]. By using the BamHI and HindIII restriction cloning, the insert of interest could be fused to the GST protein, allowing them to be more easily expressed in <I>E. coli</i>. Thus, with the help of a GST protein, Xpress is the optimal vector for the production of difficult proteins. </p>
  
 
===Source===
 
===Source===

Revision as of 00:54, 22 October 2021


Xpress: T7-Compatible Expression Vector for E. coli with A GST solubility Tag


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 33
    Illegal XbaI site found at 1908
    Illegal PstI site found at 2955
    Illegal PstI site found at 3842
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 33
    Illegal PstI site found at 2955
    Illegal PstI site found at 3842
    Illegal NotI site found at 3
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 33
    Illegal BamHI site found at 757
    Illegal XhoI site found at 912
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 33
    Illegal XbaI site found at 1908
    Illegal PstI site found at 2955
    Illegal PstI site found at 3842
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 33
    Illegal XbaI site found at 1908
    Illegal PstI site found at 2955
    Illegal PstI site found at 3842
    Illegal NgoMIV site found at 2201
    Illegal NgoMIV site found at 2598
    Illegal NgoMIV site found at 2608
    Illegal NgoMIV site found at 2734
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 3694
    Illegal SapI.rc site found at 160
    Illegal SapI.rc site found at 2705


Design Notes

Quick Screening: Screening for positive colonies following cloning is often a long and tedious task that often requires screening of many colonies. With the help of a unique feature on the Xpress vector, screening for a positive colony could be done in one single step. Xpress contains two selection markers: a kanamycin resistance gene and an ampicillin resistance gene. However, upon cloning of the insert into the appropriate spot in the multiple cloning sites, the kanamycin resistance gene will be knocked out. As such, successfully cloned colonies will only be resistant to ampicillin whereas colonies that have not been engineered properly will retain their kanamycin resistance. By streaking colonies on ampicillin and kanamycin plates, successful and unsuccessful colonies could be easily differentiated from one another as the successful colonies are expected to only grow on the ampicillin plates while all other negative colonies will show growth on both kanamycin and ampicillin plate. Thus, in this one simple step Xpress allows for fast screening of positive colonies.

Ease of Expression: E. coli has been an extremely powerful factory for producing proteins. Heterologous protein expression, however, could sometimes prove challenging. In particular, for proteins that are not suited to the natural expression conditions found in E. coli or for insoluble proteins that are difficult to produce. Xpress contains a GST tag which is a protein that is highly soluble and easily produced by E. coli. fusion of GST to heterologous proteins has proven to help with their expression as GST can act as a chaperone that helps with the proper folding of the protein [1]. By using the BamHI and HindIII restriction cloning, the insert of interest could be fused to the GST protein, allowing them to be more easily expressed in E. coli. Thus, with the help of a GST protein, Xpress is the optimal vector for the production of difficult proteins.

Source

a

References