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KillerRed is our project's star protein and the key to our bacterial cell density control system. It represents the light-sensitive element that allows the cells to receive signals from the control device.
KillerRed is a red fluorescent protein [1], meaning that by illuminating it with wavelengths from a certain portion of the visible spectrum, it re-emits light in another portion with longer (less energetic) wavelengths. Below is the absorption and emission spectra for the KillerRed protein:
The KillerRed protein absorption (left peak) and emission (right peak) spectra
Source:Detailed KillerRed description from Evrogen
From the emission and absorption spectra, we can determine that the protein absorbs in the green portion of the spectrum with a peak at 585 nm and emits in the red portion of the spectrum with a peak at 610 nm, hence the name "KillerRed".
Emitted light from bacteria is proportional to the amount of protein in the cells. This allows for measuring protein concentration in a cell culture.
The most interesting function of the protein however is that it emits ROS (Reactive Oxygen Species) when fluorescing.[1]
ROS are highly unstable and react chemically with many substrates including proteins, lipids and DNA. These reactions are oxidative and damage the affected molecules, making ROS toxic to the cell. With sufficient amounts of ROS, a cell's essential components can be damaged beyond repair, and the cell killed. Thus illuminating KillerRed-expressing cells with light in the green portion of the visible spectrum kills them, a mechanism that we use to control cell density in a culture.
In order to understand why KillerRed has its unique properties it is necessary to look at its structure. The protein is remarkably similar to other fluorescent proteins like GFP (Aequorea victoria> and dsRed (Discosoma striata), featuring a beta-barrel housing a central alpha helix with the fluorescent chromophore at its center[2]. Normally the chromophore is protected from the outside environment by the protein shell, but this isn't the case with KillerRed.
A comparison of the 3D structures of monomerix dsRed (left) and dimeric KillerRed (right)
Credits to Carpentier P., Violot S., Blanchoin L., Bourgeois D. for the KillerRed structure, and Strongin D.E., Bevis B., Khuong N., Downing M.E., Strack R.L., Sundaram K., Glick B.S., Keenan R.J. for the dsRed structure.
Source: RCSB protein database entries 2WIQ and 2VAD.
KillerRed is a 240 amino acid protein with a 3D structure similar to other fluorescent proteins, with an eleven-strand beta-barrel surrounding an alpha-helix containing the chromophore, source of the protein's fluorescence and photoxicity.
KillerRed has a DsRed-type chromophore formed with residues 67Q (glutamine), 68Y (tyrosine), and 69G (glycine), to make QYG. The corresponding coding sequence can be found at the code segment CAGTACGGC.
The interesting properties of the protein are directly related to a unique structural difference among fluorescent proteins, consisting in an open channel linking the chromophore to the environment outside the protein. According to litterature, this is the reason KillerRed is able to produce 1000-fold more reactive oxygen species compared to EGFP which is another ROS-producing fluorescent protein.[2]
KR was originally engineered from the anm2CP anthomedusa chromoprotein by individual amino acid mutations in order to obtain fluorescence and phototoxicity.[1]
This section of the wiki describes the initial construction of a prokaryotic genetic network allowing the expression of KillerRed in E. coli
The KillerRed gene was in a eukaryotic plasmid when we obtained it initially. To get expression of the protein in E. coli, it is necessary to transfer this gene into a prokaryotic plasmid. The choice of the plasmid and the genetic components inside (Promoter, RBS) is particularly important because we want to control how fast KillerRed kills the cells. To do this we must control the amount of protein in the cells. There is no literature about the effects of KillerRed on cells in low light, so we suspect KillerRed could be toxic even at low doses of light. This represents an additional factor we had to take into account during the construction of the plasmid.
Since the protein could be toxic, we want to control when KillerRed is expressed in the cells, and so we have to choose inducible promoters. In this way, even if cell growth is slowed by KillerRed we can easily measure its effects by plating the culture on agar plates and then counting the number of colonies. But to do that with some precision, and to obtain enough protein for the phototoxic effectwe could make sure that we had a significant cell population in the culture before expressing something potentially toxic. We then had two different inducible promoters to choose from
[1] M.E. Bulina et al., A genetically encoded photosensitizer, Nature Biotechnology, January 2006.
[2] Sergei Pletnev et al., Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed, The Journal of Biological Chemistry vol. 284, no. 46, pp. 32028–32039, November 13, 2009.
[3] Russell B. Vegh et al., Reactive oxygen species in photochemistry of the red fluorescent protein ‘‘Killer Red’’, Chem. Commun.,2011,47,4887–4889.
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