Difference between revisions of "Part:BBa K1217008"

Latest revision as of 06:38, 5 October 2013

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

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

@@ Line 6: / Line 6: @@
 ==Usage and Biology==
-Efficiency of this construct is compared with Two other designs  [https://parts.igem.org/Part:BBa_K1217008 BBa_K1217008] and [https://parts.igem.org/Part:BBa_K1217010 BBa_K1217010].
+Efficiency of this construct is compared with Two other designs  [https://parts.igem.org/Part:BBa_K1217003 BBa_K1217003] and [https://parts.igem.org/Part:BBa_K1217010 BBa_K1217010].
 We test the constructs' efficiency from 2 parameters: poly-P synthesis efficiency and phosphate removal efficiency from the medium.
 <gallery>
@@ Line 13: / Line 13: @@
 File:PpkEutS.png|Expected model of BBa_K1217010
 </gallery>
-==='''Expression of BBa_K1217003'''===
-We confirm the expression of PPK1 enzyme in e.coli.
-[[File:HKU13_KOppkSDS.png|400px|thumb|none|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'''===