Part:BBa_J428351

pJUMP27-1A KanR Type IIS Level 1 vector. Origin pSC101 (low copy number)

Characterisation Studies

Cambridge 2022 JUMP DV Characterisation

• Summary

As part of our Bronze - Contribution criteria, Cambridge 2022 characterised 3 JUMP DV plasmids in DH5a E. coli, from the 2022 distribution kit plate 1, against increasing concentrations of ammonium sulphate. These were of varying copy number: pSC101 (low copy no.), pBBR1 (low-med. copy no.) and pUC (high copy no.) to investigate the effect of ammonium on different copy number burden.

DV pSC101 was the first cloning vector to be created and also gave us the most interesting results so we are focusing our characterisation on this one. Its growth, compared to the other 2 DVs was less sporadic and didn't go through such a dramatic death phase. Low copy number plasmids are often used as default as they exert less burden on cells so this allows us to made a larger contribution to the iGEM and synthetic biologist community.

• Verdict





Figure 1: Fluorescence Intensity of JUMP DVs with varying [NH4]2SO4 mM

DV pSC101 (low copy no.) : Ammonium enhances fluorescence intensity up to 540mM. Despite the fluorescence rate decrease, the maximum fluorescence increases as we increase ammonium to 540mM with the cells reaching their limit at 720mM. Therefore, ammonium sulphate can be added to the growth media of cells containing pSC101 DV in order to enhance the expression of the protein of interest (in place of the sfGFP).

DV pBBR1 (low-med copy no.) : Ammonium enhances fluorescence intensity up to 360mM. Despite the fluorescence rate decrease, the maximum fluorescence increases as we increase ammonium to 360mM with the cells having lower expression at 540mM, reaching their limit at 720mM. Therefore, ammonium sulphate can be added to the growth media of cells containing pBBR1 DV in order to enhance the expression of the protein of interest (in place of the sfGFP).

DV pUC (high copy no.) : For high copy number plasmids, we can report that any ammonium concentration is detrimental to the cells expression. The greatest fluorescence was recorded with 0mM of ammonium sulphate, with decreasing expression as it was increased.

• Experience/Detailed Protocol: https://parts.igem.org/wiki/index.php?title=Part:BBa_J428351:Experience

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  DNA Sequence and Features

  
  

  Assembly Compatibility:

  • 10
    INCOMPATIBLE WITH RFC[10]

    Illegal prefix found at 3023
    Illegal suffix found at 16
    
  • 12
    INCOMPATIBLE WITH RFC[12]

    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3023
    Illegal SpeI site found at 17
    Illegal PstI site found at 31
    Illegal NotI site found at 24
    Illegal NotI site found at 3029
    
  • 21
    INCOMPATIBLE WITH RFC[21]

    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3023
    
  • 23
    INCOMPATIBLE WITH RFC[23]

    Illegal prefix found at 3023
    Illegal suffix found at 17
    
  • 25
    INCOMPATIBLE WITH RFC[25]

    Illegal prefix found at 3023
    Plasmid lacks a suffix.
    Illegal XbaI site found at 3038
    Illegal SpeI site found at 17
    Illegal PstI site found at 31
    Illegal NgoMIV site found at 1390
    
  • 1000
    INCOMPATIBLE WITH RFC[1000]

    Plasmid lacks a prefix.
    Plasmid lacks a suffix.