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P. Anzenhofer BSc. Thesis - Coggle Diagram
P. Anzenhofer BSc. Thesis
Mammalian Stability of BphPs and derived molecular sensors
Cloning of EP1 variants and remaining mammal. plasmids
Expression and degradation in Hela cells
: RePCM vs. DrPCM and respective sensors (repeat) + EP1 variants
WB
(temp.; timepoints (?); Abs: HisTag, GFP, GAPDH)
Fluo. Imaging
( + Calcium sensitivity)
Engineering BphP-based molecular sensors
Design of novel NIR- candidate arginine sensors
(
receptor
: ArtJ;
reporter
: RePCM;
entry
@ ArtJ_135
Plasmid design in SnapGene
Protein visualization in PyMol
Cloning of candidate NIR- Arg sensors
(designed by P.A. as above)
PCR of Gibson fragments
Restriction Digestion of pDUex backbone
Purification of all fragments
Ligation and bacterial transformation
DNA extraction and sequencing
Colony PCR
Bacterial overexpression of candidate NIR- Arg sensors
(cloned by P.A. as above)
Transformation on plate
Overnight pre-culture @ 37°C
Main culture growth @ 37°C
Main culture: expression induction and overnight incubation @ 22°C
Bacterial pellet collection
Purification of candidate NIR-Arg sensors
(from stored overexpressions of designs by E.R)
Pellet resuspension and cell lysis
Ni- affinity chromatography of clarified lysate
Size-exclusion chromatography of protein eluate fraction(s)
Concentration and aliquoting of protein eluate fraction(s)
Spectroscopical characterization of candidate NIR- Arg sensors
(from stored overexpressions of designs by E.R.)
Photo- and fluorescence spectroscopy
(with and /-out Arg)
Analysis of obtained spectra
Protein dual - expresion vector
Cloning of test protein gene in pDuex vector
Mammalian expression - comparison with N1 vector
Bacterial expression - comparison with pDuet vector