Composite

Part:BBa_K1217008

Designed by: Ma Tsz Shan   Group: iGEM13_Hong_Kong_HKU   (2013-09-15)
Revision as of 06:37, 5 October 2013 by ShannonMa (Talk | contribs) (→‎Usage and Biology)

Localizing polyphosphate kinase (ppk1) into native Eut microcompartment (BBa_K311004)

Under construction

Usage and Biology

Efficiency of this construct is compared with Two other designs BBa_K1217003 and BBa_K1217010. We test the constructs' efficiency from 2 parameters: poly-P synthesis efficiency and phosphate removal efficiency from the medium.

  • Expected model of BBa_K1217003

  • Expected model of BBa_K1217008

  • Expected model of BBa_K1217010

Expression of BBa_K1217003

We confirm the expression of PPK1 enzyme in e.coli.

SDS-PAGE analysis of expression of K.oralis PPK1 enzyme inside E.coli. Expression of PPK1 is induced by 0.5mM IPTG at 30 degree for 5 hours. Band marked with star is the PPK1 enzyme, the enzyme expressed is soluble.

Efficiency of poly-P synthesis and phosphate removal from environment

Experiment procedure. Wide type and blank act as a control to indicate the normal phosphate uptake of wide type e.coli and the phosphate level change along with time. Upon iptg induction, cultural samples are collected in every three hours. The cell pellet harvested is used to measure the intracellular poly-P level and the supernatant is used for phosphate quantification

Intracellular poly-P quantification

Intracellular poly-P is extracted from the cells and quantified using DAPI fluorescence measurement. DAPI is a fluorescent probe that binds DNA and poly-P. Since DNA in the sample has been removed during poly-p extraction step, it reduce nonspecific fluorescent signal due to DNA-DAPI binding. Poly-P standard curve is shown.
Intracellular Poly-P level of all samples. Blue: Wild type indicate the basal poly-P level inside wild type cell. Red: BBa_K1217003. Black: BBa_K1217010. Green:BBa_K1217008. All 3 constructs have higher poly-P amount accumulated.



Phosphate level in the medium

Supernatant medium collected at each time point is quantified using Malachite green colorimetric assay. Phosphate standard curve is shown.
Phosphate level in medium. Blue: Wild type indicate the basal phosphate removal from the medium by wild type cell. Red: BBa_K1217003. Green: BBa_K1217008. The two constructs have higher phosphate removal from the medium.


Testing

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 309



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 2631
    Illegal NotI site found at 3541
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2289
    Illegal BglII site found at 2844
    Illegal BglII site found at 3754
    Illegal XhoI site found at 2639
    Illegal XhoI site found at 3549
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3468
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3056
    Illegal SapI.rc site found at 366


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