Plasmid_Backbone

Part:BBa_J428347

Designed by: Marcos Valenzuela-Ortega   Group: 2021_Engineering_Committee   (2022-06-15)

pJUMP23-1A KanR Type IIS Level 1 vector. Origin pBBR1 (medium-copy; broad-host-range)

Characterisation Studies

Cambridge 2022 JUMP DV Characterisation

  • Summary
  • As part of our 'Bronze - Contribution' criteria, Cambridge 2022 characterised 3 JUMP DV plasmids, from the 2022 distribution kit plate 1, in DH5α E. coli against increasing concentrations of ammonium sulphate. The DVs 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 bacterial growth and expression with varying copy number burden.

  • Verdict

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

    DV pSC101 (low copy no.): Ammonium sulphate enhanced fluorescence intensity up to 540mM. Despite the fluorescence rate decrease, the maximum fluorescence increased as we increased ammonium sulphate concentration to 540mM with the cells reaching their limit at 720mM. Therefore, up to a maximum of 540mM of 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 our sfGFP).

    DV pBBR1 (low-med copy no.): Ammonium sulphate enhanced fluorescence intensity up to 360mM. Despite the fluorescence rate decrease, the maximum fluorescence increased as we increased ammonium sulphate concentration to 360mM with the cells having lower expression at 540mM and finally reaching their limit at 720mM. Therefore, up to a maximum of 360mM of 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 our 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: http://parts.igem.org/wiki/index.php?title=Part:BBa_J428351:Experience

    DNA Sequence and Features


    Assembly Compatibility:
    • 10
      INCOMPATIBLE WITH RFC[10]
      Illegal prefix found at 3081
      Illegal suffix found at 12
    • 12
      INCOMPATIBLE WITH RFC[12]
      Plasmid lacks a prefix.
      Plasmid lacks a suffix.
      Illegal EcoRI site found at 3081
      Illegal SpeI site found at 13
      Illegal PstI site found at 27
      Illegal NotI site found at 20
      Illegal NotI site found at 3087
    • 21
      INCOMPATIBLE WITH RFC[21]
      Plasmid lacks a prefix.
      Plasmid lacks a suffix.
      Illegal EcoRI site found at 3081
    • 23
      INCOMPATIBLE WITH RFC[23]
      Illegal prefix found at 3081
      Illegal suffix found at 13
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal prefix found at 3081
      Plasmid lacks a suffix.
      Illegal XbaI site found at 3096
      Illegal SpeI site found at 13
      Illegal PstI site found at 27
      Illegal NgoMIV site found at 1386
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Plasmid lacks a prefix.
      Plasmid lacks a suffix.


[edit]
Categories
//chassis/prokaryote/ecoli
//collections/jump/level1
//plasmidbackbone/assembly/typeiis
Parameters
resistancekanamycin