Coding

Part:BBa_K1184000

Designed by: Eric Pederson   Group: iGEM13_Carnegie_Mellon   (2013-09-04)

KillerRed

Crystal Structure of KillerRed (PDB ID: 3GB3) with the QYG chromophore shown2,3,4

KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of yellow-orange light (540-585 nm). KillerRed is engineered from anm2CP to be phototoxic. It has been shown that KillerRed produces superoxide radical anions by reacting with water. Superoxide reacts with the chromophore of KillerRed, causing it to become dark, which ultimately gives rise to a bleaching effect. KillerRed is spectrally similar to mRFP1 with a similar brightness. KillerRed is oligomeric and may form large aggregates in cells. Expression of KillerRed and irradiation with light may act a kill-switch for biosafety applications.

Superoxide (O2•-) is the radical anion of molecular oxygen, which can react with protein side chains and lipids to harm cells.

Superoxide radical anion

This sequence is codon optimized for mammalian cells and has 13 rare proline codons for E. coli (CCC) and one rare arginine codon (AGA). Codon optimization should be taken into consideration if large amounts of the protein are required. Expression on a high-copy plasmid has produced detectable fluorescent signal, however.


Characterization

2016 NYMU-Taipei team characterized this part with thermal stability assay.

KillerRed protein was extracted from transformed E.coli and a control group of empty vector was set to eliminate the influence of other proteins.

This test aims to show the temperature influence on KillerRed protein by measuring its fluorescence after being treated for 2 hours at the following temperatures: 70, 70.5, 71.7, 73.5, 75.5, 78.3, 81, 83.6, 86, 88, 89.5, 92.2 (degree Celsius).

Figure1: Result of heat denaturation experiment


  • It is obvious that KR contribute to most of the fluorescence.


Figure2: Curve fitting of KillerRed fluorescence


  • A sigmoidal curve was fitted to find the particular temperature K at which half of the KR protein is denatured studied by measuring the fluorescence.



Cha formula.png




a=19.42

n=36.1

K=75.79

R-square=0.998




NYMU 2016 also developed this part to construct a fungal expression cassette. (BBa_K2040121)

Figure3. The bright field and fluorescence image of WT and KR transformants under Fluorescence Microscopy Olympus BX61
  • This new device was successfully expressed in M.anisopliae




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 BsaI.rc site found at 151
    Illegal BsaI.rc site found at 442


[edit]
Categories
//biosafety
//biosafety/kill_switch
//cds/reporter/rfp
//chassis/prokaryote/ecoli
//function/celldeath
//function/reporter/fluorescence
Parameters
colorRed
controlBBa_E1010 as a negative control
emission611 nm
excitation545-585 nm
functionPhototoxicity
originEngineered from anm2CP
outputSuperoxide radical anion