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pLac-RBS-KillerRed
This BioBrick was obtained by PCR on an eukaryote vector containing the coding sequence of the KillerRed protein (generous gift of Stefan Dimitrov and Yohan Roulland, IAB Grenoble). A PCR allowed to flank it with the restriction sites KpnI and BamHI upstream and downstream. It was then ligated into the pQE30 vector(Qiagen), which contains a pLac promoter and RBS.
The KillerRed protein fits in our project in 2 ways: its fluorescence makes the transformed cells easily detectable and can be used to quantify the amount of live cells by our device. Its ROS-mediated killing is triggered by light, such that no chemical product has to be added in the medium to make it work and the process can be stopped as soon as the light is switched off.
pLac-RBS-KillerRed
This BioBrick was obtained from BBa_K1141001 using PCR to amplify the part pLac-RBS-KillerRed. An overlapping PCR was performed to remove an EcoRI restriction site from the sequence in between pLac and RBS. The sequence was then flanked with the iGEM suffix and prefix.
pLac-RBS-mCherry
This part contains the BioBricks BBa_R0010 and BBa_J06702.
It was built with standard assembly.
In the project this BioBrick was used as a negative control of KR as mCherry has similar excitation and emission peaks without being cytotoxic (very low ROS production). This BioBrick also allowed us to characterize the two BioBricks BBa_R0010 and BBa_J06702 with new data.
Green sensor-GFP
Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
This part was used in the light controlled module it has been built to characterize the green sensor.
Red sensor-RFP
Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab
This part is a negative control that does not produce as much ROS a KR.
Red Sensor-KillerRed
Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
This BioBrick takes part in the light controlled KillerRed concentration module. It is the enhanced version of BBa_K1141001 with a light sensitive promoter.
pBad-RBS-sspB
This part contains the BioBricks pBAD K206000 and sspB K174000, the RBS has been added via PCR on sspB, the rest of the BioBrick was built by standard assembly.
This BioBrick is a part of the degradation-module, it enhances the degradation rate of any protein tagged with an ''ssrA''-tag.
KillerRed is a key protein in our bacterial 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]
[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.
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