Plasmid_Backbone

Part:BBa_J428351:Experience

Designed by: Marcos Valenzuela-Ortega   Group: 2021 Engineering Committee   (2022-06-15)
Revision as of 23:52, 10 October 2022 by Sw2014 (Talk | contribs)


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Applications of BBa_J428351

Cambridge 2022 JUMP DV Characterisation

Experimental approach

JUMP DV Considerations

Out of the 3 JUMP DVs that we characterised in E. coli, we aim to focus on pSC101 as its growth, compared to the other 2 DVs, was less sporadic and didn't go through such a dramatic death phase, instead remaining at the stationary phase until the end of our experiment. Low copy number plasmids are also often used as a default as they exert less burden on cells so this allows us to made a larger contribution to the iGEM and synthetic biologist community. pSC101 being a low-copy number plasmid, will therefore exert less burden on our cells - this is in particular important for the construction and functionality of the complex 'robust perfect adaptation' circuit.

Experimental Setup & Protocol

We used a 96- well plate in the Clariostar Plate reader, changing the ammonium sulphate concentration in the growth media and the copy number plasmid in DH5a E. coli:

Figure 2: 96 Well Plate Layout
Liquid Culture Preparation
  1. Label 5 snap caps with plasmid/sample name: ‘just cells’, ‘lvl 1’, ‘pSC101’, ‘pBBR1’, ‘pUC’
  2. 2mL of EZRDM added to all 5
  3. In all but ‘just cells’, 2uL of Kan antibiotic is added
  4. Innoculate liquid culture
  5. Put snapcaps in shaking incubator for 24 hours at 30 degrees at 180rpm
Pippetting the well plate
  1. Make 720mM (filter sterilised) stock:
    14.4mL of 1000mM [NH4]2SO4 & 5.6mL EZRDM (0mM) =20mL
  2. Label 5 snapcaps with the 5 ammonium sulphate concentrations:
    720mM: 4mL 720mM ([NH4]2SO4)
    540mM: 3mL 720mM ([NH4]2SO4) & 1mL 0mM (EZRDM)
    360mM: 2mL 720mM ([NH4]2SO4) & 2mL 0mM (EZRDM)
    180mM: 1mL 720mM ([NH4]2SO4) & 3mL 0mM (EZRDM)
    0mM: 750mM: 4mL 0mM (EZRDM)
  3. Pipette up 10mL of [NH4]2SO4 using stripette and release 4ml,3ml,2ml,1ml into 720mM, 540mM, 360mM, 180mM. (leave 0mM empty)
  4. Then pipette up 10mL of EZRDM using stripette and release 4ml,3ml,2ml,1ml into 0mM, 180mM, 360mM, 540mM. (leave 720mM alone)
  5. They should all have final vol. of 4mL
  6. Get red pen to mark of wells I’ve added ammonium sulphate sol. to
  7. Get green pen to mark when cells have been added.
  8. ‘Just cells’ will be the only wells with things in without Kanamycin. So pipette 3x 200uL of each stock (into the wells with their replicates:
    ‘Just cells’ = 0,0,0,180,180,180,360,360,360,540,540,540,720,720,720.)
  9. All the stock now need Kanamycin added so 600uL is gone from each, leaving 3400uL left.
  10. Add 3.4uL Kan to all the 5 stocks - our just EZRDM with diff ammonium sulphate conc.s wells will also have Kanamycin
  11. Pipette 200uL of the respective stocks into their wells
  12. Pipette 1uL of the respective cells into their wells.
  13. Put transparent film on top of well plate and put in plate reader.




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